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Transcriptional Integration of Distinct Microbial and Nutritional Signals by the Small Intestinal Epithelium

Open AccessPublished:May 06, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.04.013

      Background & Aims

      The intestine constantly interprets and adapts to complex combinations of dietary and microbial stimuli. However, the transcriptional strategies by which the intestinal epithelium integrates these coincident sources of information remain unresolved. We recently found that microbiota colonization suppresses epithelial activity of hepatocyte nuclear factor 4 nuclear receptor transcription factors, but their integrative regulation was unknown.

      Methods

      We compared adult mice reared germ-free or conventionalized with a microbiota either fed normally or after a single high-fat meal. Preparations of unsorted jejunal intestinal epithelial cells were queried using lipidomics and genome-wide assays for RNA sequencing and ChIP sequencing for the activating histone mark H3K27ac and hepatocyte nuclear factor 4 alpha.

      Results

      Analysis of lipid classes, genes, and regulatory regions identified distinct nutritional and microbial responses but also simultaneous influence of both stimuli. H3K27ac sites preferentially increased by high-fat meal in the presence of microbes neighbor lipid anabolism and proliferation genes, were previously identified intestinal stem cell regulatory regions, and were not hepatocyte nuclear factor 4 alpha targets. In contrast, H3K27ac sites preferentially increased by high-fat meal in the absence of microbes neighbor targets of the energy homeostasis regulator peroxisome proliferator activated receptor alpha, neighbored fatty acid oxidation genes, were previously identified enterocyte regulatory regions, and were hepatocyte factor 4 alpha bound.

      Conclusions

      Hepatocyte factor 4 alpha supports a differentiated enterocyte and fatty acid oxidation program in germ-free mice, and that suppression of hepatocyte factor 4 alpha by the combination of microbes and high-fat meal may result in preferential activation of intestinal epithelial cell proliferation programs. This identifies potential transcriptional mechanisms for intestinal adaptation to multiple signals and how microbiota may modulate intestinal lipid absorption, epithelial cell renewal, and systemic energy balance.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ANOVA (analysis of variance), bp (base pair), CV (colonized), DHS (DNase hypersensitivity site), FA (fatty acid), FAO (fatty acid oxidation), GF (germ-free), GFP (green fluorescent protein), GO (Gene Ontology), HFM (high-fat meal), HNF4A (hepatocyte nuclear factor 4 alpha), IEC (intestinal epithelial cell), ISC (intestinal stem cell), LRT (likelihood ratio test), PCA (principal components analysis), PPARA (peroxisome proliferator activated receptor alpha), RNA-seq (RNA sequencing), ROI (region of interest), TF (transcription factor)
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      Collectively, this suggests HNF4A binding activity and function in IECs are relatively high in a GF context and may regulate the response to microbial and nutritional signals. However, the underlying reason for the overlap between microbial and hnf4a-regulated genes and how microbes alter HNF4A occupancy, host metabolism, and acquisition of nutrients remain unknown.
      Here we applied multiple functional genomic assays to evaluate the interaction between HFM and microbiota colonization in mouse small intestinal IECs. Entry of a HFM into the small intestine initiates a dynamic postprandial process beginning with emulsification and digestion within the lumen, leading to uptake of lipids into IECs where they are temporarily stored in cytosolic lipid droplets, oxidized as a fuel source, or exported in chylomicron lipoproteins that are distributed via the circulatory system. We focused here on a single, early postprandial time point after gavage of a complex HFM consisting of chicken egg yolk emulsion, chosen to capture transcriptional responses during the initial perception and response to a complex HFM before cell-division and cell-type changes are substantial. Collectively, our results identify the chromatin-based and transcriptional interplay between microbial and nutritional responses in the jejunal epithelium and suggest microbes may promote lipid accumulation, weight gain, and proliferation by suppressing HNF4A, PPARA, and FAO in the intestine after a HFM.

      Results

      Characterizing Intestinal Adaptation to Microbes and HFM

      By manipulating the presence of microbiota and HFM, we queried 4 conditions in the adult mouse jejunum: GF, GF+HFM, ex-GF colonized with a conventional microbiota for 2 weeks (colonized, CV), and CV+HFM (Figure 1A). Previous studies suggested that 2 hours after HFM gavage was sufficient to initiate lipid droplet accumulation and chylomicron export in IECs, but too early in the postprandial process to detect major differences in intestinal lipid transport between GF and CV mice.
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      In accord, we found that gavaging mice with HFM consisting of a chicken egg yolk emulsion labeled with BODIPY-conjugated C12 FA led to BODIPY accumulation in jejunal IECs after 2 hours (Figure 1B). Wholemount confocal microscopy of villi from mice in each +HFM condition identified an increase in epithelial BODIPY signal in CV+HFM relative to GF+HFM, which was significant on the basis of a signal per villi measurement, but not when comparing across mice (Figure 1C). This is consistent with previous reports that microbial colonization promotes lipid accumulation in IECs
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      • Leone V.
      • Chang E.B.
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      ,
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      and confirms that lipid has reached jejunal IECs by this time point. We next performed direct infusion MS/MSALL lipidomic analysis
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      on jejunal IEC preparations (see Methods) to assess potential differences in lipid content under these 4 conditions. Comparing the relative abundance of major lipid classes in the 4 conditions revealed a small significant increase of triacylglycerol in CV+HFM compared with GF+HFM, consistent with increased lipid absorption in CV (Figure 1D, Supplementary Table 1). Analysis of FA saturation within the entire neutral lipid pool (i.e., triacylglycerols, diacylglycerols, and cholesteryl esters) revealed relative increases in the abundance of saturated FA in both HFM conditions (38% increase relative to GF [P = .0051] and 53% increase relative to CV [P = .0009]) and relative decreases in polyunsaturated FA (20% decrease relative to GF [P = .0224] and 36% decrease relative to CV [P < .0001]; Figure 1E). Analysis of individual FA species within the neutral lipid pool suggested these differences were driven largely by relative increases in the abundance of FA 16:0 and 18:0 in both HFM conditions and relative decreases in FA 18:2 (Figure 1F), presumably reflecting the influx of saturated FA that predominates in chicken egg yolk.
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      Analysis of other FA species and broader lipid classes suggested other potential impacts of microbiota and HFM feeding (Figure 1F, Supplementary Table 1). Together these results establish that 2 hours of HFM feeding is sufficient to initially incorporate dietary lipids into IECs, with only minor differences in lipid content between GF and CV states at this early postprandial stage.
      Figure thumbnail gr1
      Figure 1Impact of HFM and microbes on the mouse intestine. (A) Experimental schematic highlighting microbial and nutritional conditions, genomic assays, and analysis. (B) Confocal en face images of jejunal villi 2 hours after gavage with egg yolk labeled with BODIPY C12 (red) with nuclei labeled with DAPI (blue). (C) Quantification of mean BODIPY C12 fluorescence per villus. Data points represent individual villi (27 GF+HFM villi, 42 CV+HFM villi), with open and closed circles representing villi from 2 biological replicate mice per condition. Averages and standard deviations of villi measurements are shown. Student t test showed significant differences when comparing across villi (P = .023) but not mice (P = .304). (D) Relative abundance of major lipid classes in each sample type including neutral lipids (triacylglyceride [TAG]), diacylglyceride [DAG], cholesteryl ester [CE]), phospholipids (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylserine [PS]), and polar lipids (sphingomyelin [SM], ceramide [Cer], lysophosphatidylcholine [LPC], lysophosphatidylethanolamine [LPE], hexosylceramides [HexCer]). All measurements were normalized to internal standards and are shown as fold change relative to GF. Two-way ANOVA revealed there was not a statistically significant interaction between treatment and lipid class (F33,144 = 0.8728, P = .6672). Simple main effects analysis showed that treatment did have a statistically significant effect on lipid class abundance (P = .0367). (E) Percentage of FAs detected across all neutral lipid classes (TAG, DAG, and CE) that are saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA). Two-way ANOVA revealed there was a statistically significant interaction between treatment and FA saturation group (F6,36 = 13.93, P < .0001). Simple main effects analysis showed that treatment did not have a statistically significant effect on lipid class abundance (P > .9999). (F) Percentage of FAs detected across all neutral lipid classes (TAG, DAG, and CE) with the corresponding chain length and saturation. Two-way ANOVA revealed there was a statistically significant interaction between treatment and FA type (F75,312 = 10.98, P < .0001). Simple main effects analysis showed that treatment did not have a statistically significant effect on FA abundance (P > .9999). For the data shown in (D-F), significant differences (P < .05) by post hoc Tukey multiple comparisons tests are noted for (a) GF vs CV, (b) GF+HFM vs CV+HFM, (c) GF vs GF+HFM, and (d) CV vs CV+HFM. Data are shown as average and standard deviation of 4 mice per condition. See also .

      Transcriptional Changes Integrate Microbial and Nutritional Responses in the Intestine

      RNA sequencing (RNA-seq) of unsorted jejunal IEC preparations (see Methods) showed the 4 conditions with each experimental replicate clustering based on treatment (Figure 2A). We identified hundreds of genes with significant transcriptional differences for each of the separate 4 comparisons (+CV: CV/GF and CV+HFM/GF+HFM and +HFM: GF+HFM/GF and CV+HFM/CV) (Figure 1A, Supplementary Table 2) and identified blocks of genes that were primarily impacted by either microbial or nutritional status (Figure 2B). Overlap of differential genes within both +HFM comparisons or both +CV comparisons identified substantial agreement (Figure 2C). However, we noted that numerous genes were also significantly different in response to both +CV and +HFM conditions, suggesting +CV and +HFM stimuli were both integrated at a subset of genes (Figure 2D, Supplementary Table 2). These genes include angiopoietin-like 4 (Angptl4), a secreted lipoprotein lipase inhibitor involved in partitioning triglyceride availability and lipid accumulation known to be suppressed by microbiota
      • Backhed F.
      • Ding H.
      • Wang T.
      • Hooper L.V.
      • Koh G.Y.
      • Nagy A.
      • Semenkovich C.F.
      • Gordon J.I.
      The gut microbiota as an environmental factor that regulates fat storage.
      ,
      • Backhed F.
      • Manchester J.K.
      • Semenkovich C.F.
      • Gordon J.I.
      Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.
      ,
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Camp J.G.
      • Jazwa A.L.
      • Trent C.M.
      • Rawls J.F.
      Intronic cis-regulatory modules mediate tissue-specific and microbial control of angptl4/fiaf transcription.
      and activated by fasting and high-fat diet.
      • Mattijssen F.
      • Alex S.
      • Swarts H.J.
      • Groen A.K.
      • van Schothorst E.M.
      • Kersten S.
      Angptl4 serves as an endogenous inhibitor of intestinal lipid digestion.
      ,
      • Kersten S.
      • Mandard S.
      • Tan N.S.
      • Escher P.
      • Metzger D.
      • Chambon P.
      • Gonzalez F.J.
      • Desvergne B.
      • Wahli W.
      Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.
      Angptl4 showed significant transcriptional differences in all 4 comparisons; it was elevated in GF relative to CV and further up-regulated by HFM in both microbial conditions (Figure 2D–F). Therefore, the nutritional and microbial signals that regulate gene transcription, while separable, can be additive or subtractive in their contributions to regulate transcription within the intestine.
      Figure thumbnail gr2
      Figure 2Impact of HFM and microbes on gene transcription in the mammalian intestine. (A) DESeq2 PCA of RNA-seq normalized counts in each replicate for the 4 conditions, with 4 mice per condition. Adonis permutational multivariate ANOVA of RNA-seq distance matrix; microbes: P = .002, R2 = 0.240; meal: P = .005, R2 = 0.169. (B) Heatmap of row z-scored normalized counts for genes significantly differential in at least one comparison by RNA-seq (P adjusted < .05). Examples of blocks of commonly behaving genes are marked for +HFM and +CV directional groups. (C) Pairwise comparison of maximum overlap of coincident significant RNA-seq genes (P adjusted <.05) for 8 directional significance groups shows generally coincident directionality and genes for +CV and +HFM comparisons. (D) Heatmap of example genes significantly different in both a +CV and +HFM comparison (P adjusted <.05). (E) UCSC screenshot for RNA-seq replicate levels at the mouse Angptl4 locus. (F) RNA-seq z-scored normalized counts of Angptl4, which are significant in both +CV and +HFM comparisons, show amplified relative expression in the GF+HFM condition. (G) Clustered heatmap of significance values for shared GO terms in at least 2 of 8 directional RNA-seq significance groups. (H) Scatterplot of significantly different gene log2 fold change for CV/GF and CV+HFM/GF+HFM RNA-seq (P adjusted <.05). (I) Same as (H) for GF+HFM/GF and CV+HFM/CV (P adjusted <.05).
      We next used Gene Ontology (GO) terms to functionally categorize each of the comparisons broken down into genes that were up- or down-regulated to create 8 total differential significance groups (Figure 2G, Supplementary Table 3). Surprisingly, +CV–down-regulated groups’ GO terms were clustered and similar to +HFM–up-regulated groups for terms such as metabolism of lipids and steroid metabolic process (GO term genes include Cyp27a1, Acaa2, Acox2, Acsl1, Acaa1a). Conversely, +HFM-down and +CV-up groups shared positive regulation of defense response (Nfkbiz, Duoxa2, Tnfrsf11a), suggesting for certain processes, there may be genes regulated by both nutritional and microbial inputs (Figure 2G).
      To determine whether any of the changes in gene expression varied on the basis of colonization and nutritional status, we characterized the +CV (CV/GF and CV+HFM/GF+HFM, Figure 2H) and +HFM (GF+HFM/GF and CV+HFM/CV, Figure 2I) responses separately. We observed that for most genes the magnitude and directionality of changes are consistent and positively correlated in the 2 +CV comparisons (Figure 2H). Previously characterized microbially responsive defense genes such as Saa1, Reg3g, and Tnf were up-regulated in both +CV comparisons (Figure 2H, Supplementary Table 2).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      Numerous metabolism genes were commonly down-regulated by microbes including previously characterized microbially responsive genes Angptl4, Nr0b2, Klf15, and Ces3b (Figure 2H).
      The genes that respond to HFM exposure with and without microbes followed a similar positive correlation of RNA log2 fold changes (Figure 2I). Hmgcs2, Angptl4, Creb3l3, Acot1, and Dgat2, all involved in lipid metabolism, were commonly up-regulated in both +HFM comparisons (Figure 2I). GO term analysis in +HFM-up genes identified enrichment for metabolism of lipids, peroxisome, and PPAR signaling (Supplementary Table 3). Genes down-regulated by HFM included genes involved in the unfolded protein response (Ddit3, Atf3), TFs (Nfil3, Crebl2, Klf6, Fosb), signaling (Fgf15, Fzd7, Jag1, Wnt5a), and inflammatory components (Nfkbiz, Nlr9b, Duox2, Duoxa2).

      Influence of Microbial and HFM Signals at FAO Genes

      To test more specifically for interaction between microbes and HFM and identify resultant gene expression patterns, we performed likelihood ratio test (LRT) analysis on our RNA-seq dataset across all conditions (Figure 3A, Supplementary Table 2).
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      Although only a limited number of genes (Rnf144b, Slc25a25, Grpc5a, Bmf) passed a P adjusted threshold of .05 for the LRT analysis, we proceeded to lower this cutoff to identify 621 genes with the most potential for interaction (Figure 3A and B, see Methods). Typically, putative interaction genes showed substantial increased or decreased relative expression in one condition (Figure 3C–H). In GF+HFM there was a reduction in Creb1, Acsl4, and the lipid droplet promoting Acsl3 (Figure 3E). This was in contrast to the up-regulation of FAO genes Acsl1, Acox2, Amcar, and Acad12 in GF+HFM, suggesting the FAO pathway is reduced by microbes and induced by HFM at the transcript level (Figure 3H).
      Figure thumbnail gr3
      Figure 3Characterizing putative transcriptional interaction genes. (A) Volcano plot showing interaction log2 fold change versus –log10 P value for typical (P adjusted <.05; blue) and lenient (P value <.05, >10 base mean counts; red) cutoffs identifies genes with greatest potential for interaction. In effect, the interaction log2 fold change represents the log2 ratio of (CV+HFM/CV)/(GF+HFM/GF) or (CV+HFM/GF+HFM)/(CV/GF), which are equivalent because these comparisons contain the same 4 conditions. Because negative (–, green) and positive (+, yellow) interactions are representative of the directionality of the fold change but not necessarily the nature of the interactions, these groups are also colored to help illustrate that property. (B) Heatmap of log2 FC for each comparison for interaction genes broken into the green and yellow groups. (C–H) RNA-seq z-scored normalized counts for example interacting genes, with each panel showing a different expression pattern.

      Microbial and Nutritional Stimuli Signal to Many of the Same Intestinal Regulatory Regions

      H3K27ac modification of nucleosomes flanking accessible regulatory regions is associated with activation of transcription of neighboring genes. Analysis of these sites in multiple conditions allows for robust interpretation of the genes, TFs, and pathways involved in coordinated transcriptional regulation.
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Shen Y.
      • Yue F.
      • McCleary D.F.
      • Ye Z.
      • Edsall L.
      • Kuan S.
      • Wagner U.
      • Dixon J.
      • Lee L.
      • Lobanenkov V.V.
      • Ren B.
      A map of the cis-regulatory sequences in the mouse genome.
      ,
      • Lickwar C.R.
      • Camp J.G.
      • Weiser M.
      • Cocchiaro J.L.
      • Kingsley D.M.
      • Furey T.S.
      • Sheikh S.Z.
      • Rawls J.F.
      Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.
      We identified H3K27ac levels genome-wide in jejunal IECs in the 4 conditions. As we observed in our RNA-seq (Figure 2A), H3K27ac ChIP-seq replicates grouped on the basis of the conditions along the PC1 and PC2 axis by principal components analysis (PCA); however, groups were not significantly different on the basis of HFM at this level (Figure 4A). An increased number of replicates for CV+HFM and GF+HFM relative to CV and GF improved the ability to identify differential H3K27ac CV+HFM/GF+HFM sites but also led to more significant sites relative to other comparisons (Figure 4B, Supplementary Table 4). Similar to our RNA-seq, the Angptl4 locus showed a region in intron 3 with both microbially reduced and HFM induced H3K27ac levels, with GF+HFM having the highest relative level of H3K27ac (Figure 4C). Diverse patterns of H3K27ac enrichment linked to RNA levels of neighboring genes were also found at promoter, intragenic, and intergenic regions (Figure 4D and E; two-sided Kolmogorov-Smirnov test for each comparison, P < .05). Although many sites only significantly changed in either +CV or +HFM comparisons, 1255 regulatory regions integrate signals from both microbial and HFM conditions (Figure 4F). Similar to RNA-seq, we found shared enriched GO terms across +CV and +HFM comparisons. +HFM-up and +CV-down sites were nearest to genes related to lipid metabolism, and response to other organisms was found in +HFM-down and +CV-up groups (Figures 2G and 4G, Supplementary Table 3).
      Figure thumbnail gr4
      Figure 4Identification of nutritional and microbial regulatory regions in the mammalian intestine. (A) DESeq2 PCA of H3K27ac ChIP-seq normalized counts for all replicates for each condition. Replicates represent individual mice: CV = 2, CV+HFM = 5, GF = 2, and GF+HFM = 5. Adonis permutational multivariate ANOVA of H3K27ac distance matrix; microbes: P = .002, R2 = 0.234; meal: P = .102, R2 = 0.111. (B) Venn diagram of overlap for significant H3K27ac sites for +CV and +HFM comparisons (P adjusted <.05). (C) Average H3K27ac ChIP-seq and DNase-seq signal for various conditions at the Angptl4 locus. An accessible chromatin region coincident with a characterized PPAR binding site in intron 3 was microbially suppressed and +HFM induced.
      • Mandard S.
      • Zandbergen F.
      • Tan N.S.
      • Escher P.
      • Patsouris D.
      • Koenig W.
      • Kleemann R.
      • Bakker A.
      • Veenman F.
      • Wahli W.
      • Muller M.
      • Kersten S.
      The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.
      (D) Scatterplots of RNA-seq versus H3K27ac log2 fold change for all 4 comparisons using the single nearest gene neighboring the significantly differential H3K27ac site (P adjusted <.05). Colored dots represent associated genes significant by RNA-seq in that comparison (P adjusted <.05) and associated with differential H3K27ac sites. (E) Example loci showing significantly differential H3K27ac enrichment for various comparisons including across multiple comparisons. (F) Quantification of different patterns of differential H3K27ac DNase sites (P adjusted <.05). The number of sites is enumerated above the pattern for each group that is significantly differential in either a +CV or +HFM comparison (left). One thousand two hundred fifty-five regulatory regions are responsive in both a +CV and +HFM comparison (right). (G) Coincident GREAT GO terms enrichment for 8 H3K27ac directional significance groups.
      • McLean C.Y.
      • Bristor D.
      • Hiller M.
      • Clarke S.L.
      • Schaar B.T.
      • Lowe C.B.
      • Wenger A.M.
      • Bejerano G.
      GREAT improves functional interpretation of cis-regulatory regions.
      (H) Scatterplot of average H3K27ac sites with significantly different log2 fold change window for CV/GF and CV+HFM/GF+HFM (P adjusted <.05). (I) Same as (H) for GF+HFM/GF and CV+HFM/CV. (J) Scatterplot of significant H3K27ac sites for CV/GF and CV+HFM/GF+HFM log2 FC colored by overlap with enterocyte (purple) or ISC (pink) regulatory regions. (K) Same as (J) for GF+HFM/GF versus CV+HFM/CV.

      Sites With Increased H3k27ac After HFM Behave Differently Depending on the Presence of Microbiota

      Having established the utility of our H3K27ac data, we proceeded to compare significant log2 fold change levels for H3K27ac sites differential for the 2 +CV and 2 +HFM comparisons (Figure 4H and I). Like RNA-seq (Figure 2H), we observed a general positive correlation for relative H3K27ac log2 fold change levels in the 2 +CV comparisons (Figure 4H). +CV sites with increased H3K27ac enrichment were linked to known microbial transcriptionally responsive genes such as Reg3g, Reg3B, Saa1, and Duox2.
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      The regulatory regions in both +CV-down H3K27ac comparisons neighbored lipid metabolism genes. These included FAO components such as Acaa2, Ppara, Acsl5, and Slc27a4 and TFs Klf15, Nr1h4, Zbtb16, and Id2 (Supplementary Table 3).
      • Camp J.G.
      • Frank C.L.
      • Lickwar C.R.
      • Guturu H.
      • Rube T.
      • Wenger A.M.
      • Chen J.
      • Bejerano G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.
      ,
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
      • van Baarlen P.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      However, unlike our RNA-seq analyses, we identified an unexpected separation only at sites that show increased H3K27ac enrichment after HFM, with surprisingly few sites having significance in both GF+HFM/GF-up and CV+HFM/CV-up comparisons (Figure 4I). The GF+HFM/GF-up only H3K27ac sites included lipid metabolism genes such as Acsl1, Acaa1b, Apoa1, Lipe, and Crot (Figure 4I, Supplementary Table 3). CV+HFM/CV-up only H3K27ac sites also included lipid metabolism related genes Cpt1a, Cpt2, Ppard, Dgat2, Fads1, Fads2, Fasn, and Lipg, many of which are associated with lipid anabolism
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      (Figure 4I, Supplementary Table 3). Although not represented by a particular GO term, we also noted many CV+HFM/CV-up regulatory regions neighbor genes with known roles in intestinal proliferation and intestinal stem cell (ISC) identity including Myc, Notch1, Ephb4, Ccnd1, Acsl2, and Sox9 (Figure 4I, Supplementary Table 3). To interrogate the potential impact of a change in chromatin-associated proliferation markers, we overlapped our regulatory regions with previously identified accessible regulatory sites from separately purified populations of ISCs and enterocytes.
      • Kazakevych J.
      • Sayols S.
      • Messner B.
      • Krienke C.
      • Soshnikova N.
      Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.
      We found enterocyte-associated regulatory regions had reduced enrichment, and ISC-associated regulatory regions had increased enrichment after colonization (Figure 4J). Interestingly, we saw that the separation at sites with increased enrichment after HFM resulted in CV+HFM/CV up sites overlapping with ISC-associated regulatory regions and GF+HFM/GF up sites overlapping enterocyte-associated regulatory regions (Figure 4K). This suggests that a major impact of intestinal adaptation to nutritional and microbial stimuli is alteration of enterocyte and ISC regulatory regions and that different signals may be integrated at these sites.
      Like RNA-seq, we also used LRT to characterize H3K27ac sites and found limited strong interactions. We therefore used a lenient statistical cutoff to identify 3430 H3K27ac sites with the greatest evidence of interaction (Figure 5A, Supplementary Table 5, see Methods). Comparison between H3K27ac putative interaction sites linked to genes that also had putative transcriptional interactions revealed 89.1% (197/221) were in agreement and commonly showed the same pattern across conditions (Figure 5B and C). This implies these functional processes of H3K27ac modification and gene transcription are linked, including in the regulation of divergent lipid metabolic processes and impacts on proliferation and ISCs, and are engaged differentially in response to HFM depending on microbiota colonization.
      Figure thumbnail gr5
      Figure 5Characterizing putative interaction regulatory regions. (A) Volcano plot showing interaction log2 fold change versus –log10 P value for lenient (P value <.01, >15 base mean; red) cutoffs identifies H3K27ac regulatory windows with greatest potential for interaction. Twenty thousand out of 547,000+ enriched H3K27ac windows that did not pass the lenient interaction cutoff were chosen at random to represent noninteracting sites. (B) Scatterplot comparing H3K27ac log2 interaction windows fold change linked to the RNA log2 interaction fold change for genes that also show interaction reveals a positive correlation suggesting many of these regions are causal in contributing to the transcription patterns of these genes across +CV and +HFM conditions. (C) Selected putative H3K27ac interaction windows from each cluster showing consistent patterns of H3K27ac enrichment and relative RNA levels across the 4 conditions.

      Differential Integration of Both Microbial and Nutritional Signals at the Same PPARA/FAO and ISC Regulatory Regions

      We conducted overlap analysis of significantly differential H3K27ac sites in each comparison and discovered an interesting property at the +HFM-up separating sites (Figure 4I). Rather than preferentially overlap with other +HFM sites, GF+HFM/GF-up sites overlapped with CV+HFM/GF+HFM-down sites, and CV+HFM/CV-up sites overlapped with CV+HFM/GF+HFM-up sites (Figure 6A). In fact, almost half (673/1375, 48.9%) of the +HFM-up H3K27ac sites were also microbially responsive (Figure 6B). We partitioned these H3K27ac sites into 2 groups: +HFM-up and +CV-up (red) and +HFM-up and +CV-down (blue) (Figure 6A and B). On the basis of our initial identification of proliferation and ISC genes neighboring CV+HFM/CV-up sites (Figure 4H–K), we asked whether these red sites were associated with the crypt-villus and proliferation-differentiation axis. Using a published dataset,
      • Kazakevych J.
      • Sayols S.
      • Messner B.
      • Krienke C.
      • Soshnikova N.
      Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.
      we found that the +HFM-up and +CV-up H3K27ac sites were rarely accessible solely in enterocytes and instead were frequently enriched only in ISCs, whereas the +HFM-up and +CV-down group frequently contained H3K27ac sites that were enriched in enterocytes (Figure 6C). +HFM-up and +CV-up (red) sites were enriched in processes including regulation of the immune system (Myc, Mmp14, Pglyrp1, and Stat3), regulation of epithelial cell differentiation (Arntl, Mycn, Pax6, and Dmbt1), and Notch signaling (Hes7, Notch1, Notch2, and Sox9) (Figure 6D and E, Supplementary Table 3). We saw limited enrichment for terms involved in catabolism, with the notable exceptions of the glycolysis enzyme Hk2 and limited FAO components Cpt1a, Cpt2, Ppard, and Prdm16. Several metabolic genes leading to lipid synthesis Fads1, Fads2, Fasn, Dgat2, and Acacb were only present in this +HFM-up +CV-up group, with Acacb thought to inhibit FAO in favor of lipid anabolism (Figure 6E, Supplementary Tables 3 and 4).
      • Abu-Elheiga L.
      • Matzuk M.M.
      • Abo-Hashema K.A.
      • Wakil S.J.
      Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2.
      ,
      • Abu-Elheiga L.
      • Oh W.
      • Kordari P.
      • Wakil S.J.
      Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets.
      Collectively, these +HFM-up and +CV-up (red) sites and their associated genes are indicative of a relative surplus of energy, lipogenesis, and proliferation in CV+HFM (Figure 6E).
      Figure thumbnail gr6
      Figure 6Microbial and nutritional stimuli signal to many of the same intestinal regulatory regions. (A) Pairwise comparison of maximum overlap of coincident significant H3K27ac sites (P adjusted <.05) for 8 directional significance groups identifies microbially responsive regulatory regions with CV+HFM/CV-up also being CV+HFM/GF+HFM-up (red) and GF+HFM/GF-up also being CV+HFM/GF+HFM-down (blue). (B) Heatmap of differential comparisons (P adjusted <.05) for all +HFM-up sites shows the proportion that is also microbially responsive. (C) Pie charts for red and blue +HFM-up groups that show the proportion that overlap with previously characterized enterocyte and ISC regulatory regions.
      • Kazakevych J.
      • Sayols S.
      • Messner B.
      • Krienke C.
      • Soshnikova N.
      Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.
      (D) GREAT GO term enrichment for red and blue +HFM-up H3K27ac groups. (E) Heatmap of example red +HFM-up and +CV-up H3K27ac sites and their linked gene’s RNA-seq log2 fold change, including many loci associated with ISCs and proliferation. (F) Different combinations of differential H3K27ac sites that are both +HFM and +CV responsive show that only red sites that are +HFM-up and +CV-up are linked to genes that are preferentially expressed in ISCs relative to enterocytes. (G) Heatmap of example blue +HFM-up H3K27ac sites and their linked gene’s RNA-seq log2 fold change. Blue asterisk marks Ppara regulatory region that is characterized in . (H) Different combinations of differential H3K27ac sites that are both +HFM and +CV responsive show that only blue sites that are +HFM-up and +CV-down are linked to genes that are activated by PPARA. (I) Scatterplot of genes that are significantly differential after PPARA activation and in GF+HFM/GF show a positive correlation. (J) Scatterplot of genes that are significantly differential after PPARA activation and in CV/GF show a negative correlation. (K) Scatterplot of log2 fold change levels for genes significant (<.005 P adjusted) in 24-hour fast/ad libitum versus PPARA activated genes.
      • van den Bosch H.M.
      • Bunger M.
      • de Groot P.J.
      • van der Meijde J.
      • Hooiveld G.J.
      • Muller M.
      Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting.
      (L) Scatterplot of log2 fold change levels for genes significant (<.005 P adjusted) in 24-hour fast/ad libitum versus GF+HFM/GF shows many of the same PPARA targets are activated by fasting and HFM.
      • van den Bosch H.M.
      • Bunger M.
      • de Groot P.J.
      • van der Meijde J.
      • Hooiveld G.J.
      • Muller M.
      Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting.
      We then looked at different combinations of H3K27ac sites that responded to both microbial and nutritional signals and the expression of their neighboring genes in a previously published dataset comparing small intestinal enterocytes and ISCs (Figure 6F).
      • Kazakevych J.
      • Sayols S.
      • Messner B.
      • Krienke C.
      • Soshnikova N.
      Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.
      The +HFM-up and +CV-up (red) sites were linked to genes expressed preferentially in ISCs relative to enterocytes (Figure 6F). Importantly, including +HFM-up and +CV-down (blue), no other groups of sites that responded to both +HFM and +CV signals were associated with ISC and enterocyte progenitor genes (Figure 6F).
      In contrast, genes neighboring H3K27ac sites that were +HFM-up and +CV-down (blue) (Figure 6B) were enriched for lipid metabolism GO terms such as cellular lipid catabolic process and FAO (Figure 6D, Supplementary Table 3). The GO terms included genes Angptl4, Acaa2, and Acsl5 and the gluconeogenic regulator Pck1, which generally had similar patterns at the level of RNA-seq and H3K27ac (Figure 6G). In addition, a regulatory region at the FAO regulator, Ppara, was in the +HFM-up and +CV-down group (Figure 6G). FAO genes and other genes associated with energy production are transcriptionally activated by the lipid-liganded PPARA to facilitate the production of adenosine triphosphate from lipids.
      • Dubois V.
      • Eeckhoute J.
      • Lefebvre P.
      • Staels B.
      Distinct but complementary contributions of PPAR isotypes to energy homeostasis.
      Only +HFM-up and +CV-down H3K27ac sites were substantially linked to published jejunal PPARA target genes (Figure 6H).
      • Bunger M.
      • van den Bosch H.M.
      • van der Meijde J.
      • Kersten S.
      • Hooiveld G.J.
      • Muller M.
      Genome-wide analysis of PPARalpha activation in murine small intestine.
      Consistent with this finding, comparing expression levels after PPARA activation versus HFM response or microbial response revealed a positive and negative correlation, respectively (Figure 6I and J). In addition, many of the same genes induced by HFM were also induced by fasting (Figure 6K and L).
      • van den Bosch H.M.
      • Bunger M.
      • de Groot P.J.
      • van der Meijde J.
      • Hooiveld G.J.
      • Muller M.
      Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting.
      We speculated that PPARA may be elevated in GF and that this may help explain why GF+HFM results in relatively high activation of PPARA genes relative to CV+HFM and how PPARA may integrate nutritional and microbial signals. In accord, we identified increased intestinal PPARA protein levels in GF by immunofluorescence (Figure 7A and B).
      Figure thumbnail gr7
      Figure 7Characterization of the putative PPARA regulatory region that responds to microbes and HFM. (A) PPARA immunofluorescence of small intestinal villi (red) in GF and CV mice fed ad libitum. (B) Quantification of IEC PPARA nuclear fluorescence for the crypt and villus identifies higher nuclear fluorescence in GF mice. ∗P value ≤.05, ∗∗P value ≤.01, and ∗∗∗P value ≤.001; two-way ANOVA; n = 4 per condition. (C) Exploded view of H3K27ac region at mouse Ppara locus from G that is +HFM-up and +CV-down showing signal across conditions for H3K27ac, HNF4A, and accessible chromatin for jejunum and numerous other tissues. (D) Putative TF motifs at the mouse Ppara regulatory region include multiple nuclear receptor sites, including PPARE. (E) Transgenic Tg(Mmu.Ppara:GFP) zebrafish at 6 days post-fertilization (dpf) with the mouse Ppara regulatory region upstream of a mouse cFos minimal promoter driving GFP shows expression largely limited to the anterior intestine. E' boxed inset shows a confocal cross section confirming the signal is specific to IECs. (F) Quantitative real-time polymerase chain reaction of a 4 condition experiment using whole 6 dpf Tg(Mmu.Ppara:GFP) zebrafish shows similar responses to colonization and HFM for pparaa and gfp. Significance calls for colonization, nutritional, and interaction based on 2-factor ANOVA: ∗P value ≤.05, ∗∗P value ≤.01, and ∗∗∗∗P value ≤.0001. Ten to 20 larvae per replicates; 11-12 replicates per condition. (G) FAO and ISC associated genes showing particular expression in IECs from GF+HFM and CV+HFM mice, respectively ().

      Mouse Ppara Enhancer Shows Evidence of Conserved Intestinal Expression and Microbial and HFM Responsiveness in Transgenic Zebrafish

      We hypothesized that the Ppara H3K27ac enhancer we identified may be responsible for regulating Ppara in IECs and capable of regulating Ppara expression in response to microbes and HFM (Figure 6G). The Ppara enhancer is bound by HNF4A, displays accessibility largely restricted to adult digestive tissues, and contains a PPARE and other nuclear receptor binding sites (Figure 7C and D). Stable transgenic zebrafish expressing green fluorescent protein (GFP) under control of the Ppara mouse enhancer drove expression largely in IECs (Figure 7E). We found that as in mouse, both zebrafish pparaa and GFP were down-regulated after colonization (Figure 7F). However, the GFP reporter or zebrafish pparaa was instead concordantly reduced by HFM after 6 hours in zebrafish, suggesting potentially conserved regulation but lacking induction by HFM at this time point.
      • Semova I.
      • Carten J.D.
      • Stombaugh J.
      • Mackey L.C.
      • Knight R.
      • Farber S.A.
      • Rawls J.F.
      Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish.
      ,
      • Zeituni E.M.
      • Wilson M.H.
      • Zheng X.
      • Iglesias P.A.
      • Sepanski M.A.
      • Siddiqi M.A.
      • Anderson J.L.
      • Zheng Y.
      • Farber S.A.
      Endoplasmic reticulum lipid flux influences enterocyte nuclear morphology and lipid-dependent transcriptional responses.
      Collectively, this suggests that HFM preferentially influences H3K27ac sites around genes involved in ISCs and proliferation with microbes and PPARA signaling and FAO without microbes in mouse. This effect can also be seen at key FAO and ISC markers at the level of RNA (Figure 7G).
      Other regulatory region and gene groups that respond differentially to microbes and HFM are likely important. A subset of genes were linked to H3K27ac sites that are differentially enriched in +HFM-down as well as either +CV-down or +CV-up comparisons with similar RNA-seq expression patterns. For example, the circadian-regulated lipid transporter CD36
      • Kuang Z.
      • Wang Y.
      • Li Y.
      • Ye C.
      • Ruhn K.A.
      • Behrendt C.L.
      • Olson E.N.
      • Hooper L.V.
      The intestinal microbiota programs diurnal rhythms in host metabolism through histone deacetylase 3.
      is thought to be a PPARA-activated gene; however, we found both H3K27ac and RNA-seq levels at Cd36 were reduced by colonization and HFM in our dataset (Supplementary Tables 2 and 4).
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
      • van Baarlen P.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      These differentially expressed genes may be regulated by TFs, of which many are themselves responsive to both microbes and HFM at the transcript level (Figure 8A).
      Figure thumbnail gr8
      Figure 8Enrichment of TF motifs implies HNF4A distinguishes sites that are differential between CV+HFM and GF+HFM. (A) Heatmap of TFs that are significantly different in at least one +HFM and one +CV comparison by RNA-seq. (B) Motif enrichment at DNase sites linked to +CV H3k27ac significance groups (P adjusted <.05) comparing CV/GF and CV+HFM/GF+HFM sites. For each significance +CV group the reciprocal direction is used as the background (e.g., CV/GF-up input versus CV/GF-down background). The –log10 P values are plotted for motif enrichment. Both directions are plotted on the same axis with each analysis separated by colored arrows. HNF4A motif (green asterisk) was not differentially enriched between CV/GF directional H3K27ac sites but was substantially enriched in CV+HFM/GF+HFM-down sites relative to CV+HFM/GF+HFM-up sites. (C) Same as (B) for GF+HFM/GF versus CV+HFM/CV. (D) Clustering of enrichment motif score (–log10 P value) for TF motifs that are present in multiple comparisons for 8 directional significance groups. Data are shared with (B) and (C). (E) Example TF motif enrichment patterns including those coincident with red (+HFM-up and +CV-up) and blue (+HFM-up and +CV-down) H3K27ac sites.

      HNF4A Motif Is Differentially Enriched in Microbially Suppressed H3k27ac Sites Only in the Presence of HFM

      We next used TF motif enrichment within differential H3K27ac sites to find potential transcriptional regulators in the 4 conditions (Figure 8B–E). Common motifs within +CV-up sites include established microbially responsive transcription factors such as nuclear factor kappa B, IRF, and STAT (Figure 8B). We previously showed HNF4A motifs are not significantly differentially enriched between CV/GF groups (Figure 8B).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      Surprisingly, in the presence of HFM, the HNF4A motif was enriched at sites that have reduced H3K27ac with a microbiota (CV+HFM/GF+HFM-down). We clustered motifs for each comparison to identify patterns of their enrichment in +CV and +HFM conditions (Figure 8D and E). +HFM-up and +CV-up comparisons included several motifs associated with proliferation including E2F3, ASCL1, and cMYC. Both PPAR and HNF4A showed motif enrichment in +HFM-up and +CV-down comparisons, suggesting they may integrate microbial and HFM signals associated with the blue H3K27ac sites group (Figure 8B–E).

      The Association of Hnf4a With the Response to Microbes Is Due to Its Impact on Enterocyte Differentiation and the Crypt-Villus Axis

      To understand how HNF4A binding contributes to transcriptional integration of microbial and nutritional signals, we performed HNF4A ChIP-seq in the 4 conditions. HNF4A occupancy in the intestine is known to be reduced at binding sites in the CV condition.
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      PCA showed that unlike RNA-seq and H3K27ac, HNF4A does not distinguish the 4 conditions along PC1 and PC2, with CV datasets segregating along PC1 (Figure 9A). Consistent with this, we saw the number of HNF4A binding sites increase in the CV+HFM condition relative to CV (Figure 9B, Supplementary Table 6). Differential HNF4A binding analysis identified distinct changes, with +HFM generally increasing HNF4A occupancy and +CV generally decreasing HNF4A occupancy (Figure 9C–I). Although provocative, this behavior appeared to be consistent at most sites genome-wide, limiting our ability to interpret meaningful biological differences in HNF4A occupancy levels from individual sites (Figure 9C, H, and I). We instead used the relatively stable location of HNF4A binding sites across conditions. HNF4A binding sites are numerous in the intestine, with typically 15,000–30,000+ binding sites in IECs.
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Qin Y.
      • Roberts J.D.
      • Grimm S.A.
      • Lih F.B.
      • Deterding L.J.
      • Li R.
      • Chrysovergis K.
      • Wade P.A.
      An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression.
      ,
      • Verzi M.P.
      • Shin H.
      • He H.H.
      • Sulahian R.
      • Meyer C.A.
      • Montgomery R.K.
      • Fleet J.C.
      • Brown M.
      • Liu X.S.
      • Shivdasani R.A.
      Differentiation-specific histone modifications reveal dynamic chromatin interactions and partners for the intestinal transcription factor CDX2.
      ,
      • Chahar S.
      • Gandhi V.
      • Yu S.
      • Desai K.
      • Cowper-Sal-lari R.
      • Kim Y.
      • Perekatt A.O.
      • Kumar N.
      • Thackray J.K.
      • Musolf A.
      • Kumar N.
      • Hoffman A.
      • Londono D.
      • Vazquez B.N.
      • Serrano L.
      • Shin H.
      • Lupien M.
      • Gao N.
      • Verzi M.P.
      Chromatin profiling reveals regulatory network shifts and a protective role for hepatocyte nuclear factor 4alpha during colitis.
      However, transcription level changes in Hnf4aΔIEC mice have been shown to be correlated with not just binary presence or absence but the cumulative number of neighboring HNF4A binding sites for a particular gene.
      • Chahar S.
      • Gandhi V.
      • Yu S.
      • Desai K.
      • Cowper-Sal-lari R.
      • Kim Y.
      • Perekatt A.O.
      • Kumar N.
      • Thackray J.K.
      • Musolf A.
      • Kumar N.
      • Hoffman A.
      • Londono D.
      • Vazquez B.N.
      • Serrano L.
      • Shin H.
      • Lupien M.
      • Gao N.
      • Verzi M.P.
      Chromatin profiling reveals regulatory network shifts and a protective role for hepatocyte nuclear factor 4alpha during colitis.
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      We identify a similar correlation with gene expression levels and neighboring HNF4A binding site number (Figure 9J).
      Figure thumbnail gr9
      Figure 9HNF4A’s role in promoting IEC differentiation explains correlation between Hnf4a loss and microbially responsive genes. (A) DESeq2 PCA of HNF4A ChIP-seq normalized counts shows the CV condition deviates from all other conditions. Replicates represent individual mice: CV = 3, CV+HFM = 4, GF = 3, and GF+HFM = 4. Adonis permutational multivariate ANOVA of HNF4A distance matrix; microbes: P = .004, R2 = 0 .158; meal: P = .136, R2 = 0.094. (B) HNF4A peak numbers across GF, GF+HFM, CV, and CV+HFM as well as merged across all replicates and conditions (false discovery rate <0.05). (C) Clustered heatmap of HNF4A binding sites with peaks called in at least 2 replicates compared with log2 fold change for each of the 4 comparisons. (D–G) UCSC screenshot of HNF4A binding sites that are significantly differential, corresponding to (H) and (I) at the Apoa1 (D), Acaa2 (E), Mapk6 (F), and Acot12 (G) loci. (H) Scatterplot of significantly different log2 fold change for CV/GF and CV+HFM/GF+HFM HNF4A occupancy (false discovery rate <0.05). (I) Same as (H) for GF+HFM/GF and CV+HFM/CV HNF4A occupancy. (J) Genes ordered by the number of neighboring HNF4A binding sites show a positive correlation with gene expression level. (K) Scatterplot comparison between significantly differential microbial responsive genes and Hnf4aΔIEC/WT in the jejunum.
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      Genes with more than 10 neighboring HNF4A sites are indicated (dark blue). (L) Scatterplot comparing genes that have significantly differential expression in Hnf4aΔIEC/WT colon
      • Darsigny M.
      • Babeu J.P.
      • Dupuis A.A.
      • Furth E.E.
      • Seidman E.G.
      • Levy E.
      • Verdu E.F.
      • Gendron F.P.
      • Boudreau F.
      Loss of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic inflammation resembling inflammatory bowel disease in mice.
      versus CV/GF colon.
      • Camp J.G.
      • Frank C.L.
      • Lickwar C.R.
      • Guturu H.
      • Rube T.
      • Wenger A.M.
      • Chen J.
      • Bejerano G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.
      (M) Scatterplot of significant CV/GF log2 fold change RNA-seq levels versus Hnf4aΔIEC/WT RNA levels for genes in the Defense Response GO term are not frequently linked to numerous HNF4A binding sites. (N) Percentage of significant CV/GF genes per HNF4A binding site group that are significantly down-regulated for different binding site group bins. (O) Scatterplot comparing small intestine RNA-seq log2 fold change for enterocyte/enterocyte-progenitor versus Hnf4aΔIEC/WT. 10+ HNF4A binding sites/targets (blue) contribute directly and indirectly to FAO genes (yellow) activation preferentially in enterocytes.
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      ,
      • Kim T.H.
      • Li F.
      • Ferreiro-Neira I.
      • Ho L.L.
      • Luyten A.
      • Nalapareddy K.
      • Long H.
      • Verzi M.
      • Shivdasani R.A.
      Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity.
       (P) Comparison of genes that are preferentially expressed in 5 compartments along the crypt-villus axis for various Hnf4 deletion mutants in the intestine.
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      ,
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      ,
      • Darsigny M.
      • Babeu J.P.
      • Dupuis A.A.
      • Furth E.E.
      • Seidman E.G.
      • Levy E.
      • Verdu E.F.
      • Gendron F.P.
      • Boudreau F.
      Loss of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic inflammation resembling inflammatory bowel disease in mice.
      ,
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      (Q) Average number of HNF4A binding sites per gene based on crypt-villus compartment groups.
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      (R) Scatterplot comparing jejunal microbial response (CV/GF) to Hnf4aΔIEC/WT in jejunum colored for genes preferentially expressed in the crypt (red) and villus tip (blue).
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      ,
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      (S) RNA-seq log2 fold change for small intestinal enterocyte/ISC RNA levels for groups of significantly differential down (blue arrow) and up (red arrow) genes from numerous published datasets showed a common impact of Hnf4aΔIEC/WT,
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      Hnf4agDKO/WT,
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      CV/GF in sorted ISCs,
      • Peck B.C.
      • Mah A.T.
      • Pitman W.A.
      • Ding S.
      • Lund P.K.
      • Sethupathy P.
      Functional transcriptomics in diverse intestinal epithelial cell types reveals robust microRNA sensitivity in intestinal stem cells to microbial status.
      and human ileal Crohn’s
      • Haberman Y.
      • Tickle T.L.
      • Dexheimer P.J.
      • Kim M.O.
      • Tang D.
      • Karns R.
      • Baldassano R.N.
      • Noe J.D.
      • Rosh J.
      • Markowitz J.
      • Heyman M.B.
      • Griffiths A.M.
      • Crandall W.V.
      • Mack D.R.
      • Baker S.S.
      • Huttenhower C.
      • Keljo D.J.
      • Hyams J.S.
      • Kugathasan S.
      • Walters T.D.
      • Aronow B.
      • Xavier R.J.
      • Gevers D.
      • Denson L.A.
      Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature.
      on the crypt-villus/proliferation-differentiation axis.
      • Kazakevych J.
      • Sayols S.
      • Messner B.
      • Krienke C.
      • Soshnikova N.
      Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.
      FAO genes are also more highly expressed in enterocytes versus ISCs (). Yellow bars refer to the average enterocyte/ISC log2 fold change RNA levels for each group. Gray bar represents the average for all genes except FAO genes. (T) Scatterplot comparing PPARA-activated genes versus Hnf4aΔIEC/WT RNA levels in mouse jejunum identifies that PPARA targets and FAO genes are commonly reduced in Hnf4aΔIEC.
      Using a previously published dataset of mice lacking Hnf4a in jejunal IECs (Hnf4aΔIEC),
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      we found that HNF4A-activated genes are also more highly expressed in the GF condition relative to CV (Figure 9K). That suggests the relationship found in zebrafish between the HNF4A regulon and microbially responsive genes is conserved in mouse jejunum, with a similar relationship also seen in mouse colon (Figure 9L).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      We also saw that genes up-regulated in Hnf4aΔIEC mice are correlated with those up-regulated after colonization (Figure 9K), with defense response related genes such as Saa1 being consistently up-regulated by Hnf4a loss and presence of microbes across species (Figure 9K and M).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      We found that direct target genes with abundant neighboring HNF4A binding sites (10+ binding sites) are more likely to lose expression in both Hnf4aΔIEC and after colonization with microbes (Figure 9K). Conversely, by this metric, genes induced by microbial colonization and in Hnf4aΔIEC are less frequently direct HNF4A targets, suggesting inflammatory induction after HNF4A loss is not due to derepression of HNF4A targets (Figure 9K, M, and N).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Darsigny M.
      • Babeu J.P.
      • Dupuis A.A.
      • Furth E.E.
      • Seidman E.G.
      • Levy E.
      • Verdu E.F.
      • Gendron F.P.
      • Boudreau F.
      Loss of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic inflammation resembling inflammatory bowel disease in mice.
      Importantly, even at the remaining loci with little or no evidence of being a direct HNF4A target, a correlation between Hnf4aΔIEC and CV/GF expression levels remained for both up- and down-regulated genes (Figure 9K). This implicates direct, indirect, or non-canonical HNF4A functions as contributors to the correlation between HNF4A-regulated and microbially regulated genes in the jejunum. HNF4A function in response to microbiota may also be distributed across multiple regulatory regions for a particular gene.
      • Chen L.
      • Luo S.
      • Dupre A.
      • Vasoya R.P.
      • Parthasarathy A.
      • Aita R.
      • Malhotra R.
      • Hur J.
      • Toke N.H.
      • Chiles E.
      • Yang M.
      • Cao W.
      • Flores J.
      • Ellison C.E.
      • Gao N.
      • Sahota A.
      • Su X.
      • Bonder E.M.
      • Verzi M.P.
      The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac.

      Microbial and Nutritional Stimuli Influence the Crypt-Villus Axis Leading to Differential Utilization of HNF4A and Differentiation Programs

      We investigated whether more global changes in cell identity or cellular programs explain the relationship between HNF4A and microbial colonization. HNF4A and HNF4G initially were identified as positive regulators of enterocyte differentiation with reduced HNF4A activity in proliferating cells.
      • Verzi M.P.
      • Shin H.
      • He H.H.
      • Sulahian R.
      • Meyer C.A.
      • Montgomery R.K.
      • Fleet J.C.
      • Brown M.
      • Liu X.S.
      • Shivdasani R.A.
      Differentiation-specific histone modifications reveal dynamic chromatin interactions and partners for the intestinal transcription factor CDX2.
      ,
      • Babeu J.P.
      • Darsigny M.
      • Lussier C.R.
      • Boudreau F.
      Hepatocyte nuclear factor 4alpha contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation.
      ,
      • Cattin A.L.
      • Le Beyec J.
      • Barreau F.
      • Saint-Just S.
      • Houllier A.
      • Gonzalez F.J.
      • Robine S.
      • Pincon-Raymond M.
      • Cardot P.
      • Lacasa M.
      • Ribeiro A.
      Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and barrier function of the adult intestinal epithelium.
      HNF4A and HNF4G are believed to work together to promote brush border genes and in regulating FAO in ISCs.
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      ,
      • Chen L.
      • Luo S.
      • Dupre A.
      • Vasoya R.P.
      • Parthasarathy A.
      • Aita R.
      • Malhotra R.
      • Hur J.
      • Toke N.H.
      • Chiles E.
      • Yang M.
      • Cao W.
      • Flores J.
      • Ellison C.E.
      • Gao N.
      • Sahota A.
      • Su X.
      • Bonder E.M.
      • Verzi M.P.
      The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac.
      Using previously published datasets, we find genes that are preferentially expressed in terminally differentiated enterocytes relative to enterocyte progenitors are negatively correlated with expression in Hnf4aΔIEC mouse jejunum (Figure 9O).
      • Kim T.H.
      • Li F.
      • Ferreiro-Neira I.
      • Ho L.L.
      • Luyten A.
      • Nalapareddy K.
      • Long H.
      • Verzi M.
      • Shivdasani R.A.
      Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity.
      We also find loss of Hnf4a resulted in increased expression of crypt genes and a decrease of genes expressed in the differentiating villus in the jejunum, colon, or the simultaneous loss of Hnf4a and Hnf4g in duodenum (Figure 9P).
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      ,
      • Darsigny M.
      • Babeu J.P.
      • Dupuis A.A.
      • Furth E.E.
      • Seidman E.G.
      • Levy E.
      • Verdu E.F.
      • Gendron F.P.
      • Boudreau F.
      Loss of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic inflammation resembling inflammatory bowel disease in mice.
      ,
      • San Roman A.K.
      • Aronson B.E.
      • Krasinski S.D.
      • Shivdasani R.A.
      • Verzi M.P.
      Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2.
      ,
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      HNF4A binding site number per gene also showed enrichment in villus-expressed genes with limited HNF4A binding at genes preferentially expressed in the crypt (Figure 9Q).
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      To simultaneously interrogate the impact of HNF4A and microbes on the crypt-villus axis, we overlaid the preferential position of a gene’s expression in 5 zones along the crypt-villus axis relative to microbial response and Hnf4a dependence (Figure 9R).
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      Genes that were both microbially suppressed and lose expression in Hnf4aΔIEC were preferentially expressed in the differentiated villus. In contrast, microbially activated genes and those with increased expression in Hnf4aΔIEC coincidentally were preferentially expressed in the crypt, directly implicating HNF4A and the response to microbes in regulating differences in the crypt-villus axis and enterocyte differentiation (Figure 9R).
      In support of the hypothesis that microbial responses impact the underlying differentiation status of IECs, we queried a published RNA-seq dataset of sorted jejunal ISCs in separate GF and CV conditions. We not only observed the previously identified increased expression of proliferation and ISC genes after microbial exposure but also significantly reduced expression of genes preferentially expressed in enterocytes versus ISCs (Figure 9S).
      • Peck B.C.
      • Mah A.T.
      • Pitman W.A.
      • Ding S.
      • Lund P.K.
      • Sethupathy P.
      Functional transcriptomics in diverse intestinal epithelial cell types reveals robust microRNA sensitivity in intestinal stem cells to microbial status.
      Therefore, even when analysis is constrained solely to a population of ISCs, the ISC-enterocyte differentiation axis can be influenced by microbes and is unlikely to be driven solely by differences in IEC subtype composition between conditions in our experiments. This differential expression of genes along the crypt-villus axis may also explain the relationship between inflammatory phenotypes and HNF4A observed in human Crohn’s disease (Figure 9S).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Haberman Y.
      • Tickle T.L.
      • Dexheimer P.J.
      • Kim M.O.
      • Tang D.
      • Karns R.
      • Baldassano R.N.
      • Noe J.D.
      • Rosh J.
      • Markowitz J.
      • Heyman M.B.
      • Griffiths A.M.
      • Crandall W.V.
      • Mack D.R.
      • Baker S.S.
      • Huttenhower C.
      • Keljo D.J.
      • Hyams J.S.
      • Kugathasan S.
      • Walters T.D.
      • Aronow B.
      • Xavier R.J.
      • Gevers D.
      • Denson L.A.
      Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature.
      We speculated that HNF4A may promote preferential expression of FAO genes in differentiated enterocytes in the GF and GF+HFM conditions relative to CV+HFM. We found that FAO genes are preferentially expressed in enterocytes versus ISCs and enterocyte versus enterocyte progenitors in normal chow-fed conditions (Figure 9O and S, Supplementary Table 7). Similarly, loss of Hnf4a in jejunum results in loss of expression of FAO genes, PPARA targets generally, and Ppara itself (Figure 9T).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Bunger M.
      • van den Bosch H.M.
      • van der Meijde J.
      • Kersten S.
      • Hooiveld G.J.
      • Muller M.
      Genome-wide analysis of PPARalpha activation in murine small intestine.
      ,
      • San Roman A.K.
      • Aronson B.E.
      • Krasinski S.D.
      • Shivdasani R.A.
      • Verzi M.P.
      Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2.
      As a result, some of the capacity for FAO in the jejunum relies on HNF4A potentiating an enterocyte differentiation program upstream of Ppara and FAO targets.
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.

      Condition Specific H3k27ac Changes Are Restricted to Enterocyte- and ISC-Specific Regulatory Regions and Impacted by HNF4A Binding

      Because 2 hours after a HFM is likely too soon to induce major changes in IEC cell-type abundance,
      • Cheng H.
      • Leblond C.P.
      Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine: V—unitarian theory of the origin of the four epithelial cell types.
      ,
      • Al-Dewachi H.S.
      • Wright N.A.
      • Appleton D.R.
      • Watson A.J.
      Cell population kinetics in the mouse jejunal crypt.
      the enrichment of HNF4A motifs in GF+HFM (Figure 8B–E) and differential utilization of enterocyte and ISC regulatory regions in +HFM-up conditions (Figure 4J and K, Figure 6C) may be due to a global change in the utilization of the enterocyte-differentiation transcriptional program within the larger IEC population (Figure 9S). We generated a limited intestinal genomics database using a published dataset of accessible chromatin mapping of ISCs, proliferating transit-amplifying cells, enteroendocrine cells, and enterocytes using a SRY-Box Transcription Factor 9 (SOX9) sorting strategy from adult mouse small intestine to identify simple patterns in IEC accessibility (Figure 10A).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Raab J.R.
      • Tulasi D.Y.
      • Wager K.E.
      • Morowitz J.M.
      • Magness S.T.
      • Gracz A.D.
      Quantitative classification of chromatin dynamics reveals regulators of intestinal stem cell differentiation.
      To test the impact of the ISC-enterocyte differentiation axis, we grouped enterocyte-accessible regions that were also accessible across all other IEC subtype populations (pan-accessible, includes ISC), sites containing enterocyte accessibility and at least one other IEC subtype (enterocyte-restricted), and enterocyte-specific accessible sites (enterocyte-specific) (Figure 10A). We similarly grouped ISC-specific and ISC-restricted sites. Using DNase hypersensitivity site (DHS) accessibility from multiple cell types and our published data from colon and ileum, we identified that pan-accessible sites are frequently accessible constitutively across all cell types (Figure 10A).
      • Camp J.G.
      • Frank C.L.
      • Lickwar C.R.
      • Guturu H.
      • Rube T.
      • Wenger A.M.
      • Chen J.
      • Bejerano G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.
      ,
      • Mouse E.C.
      • Stamatoyannopoulos J.A.
      • Snyder M.
      • Hardison R.
      • Ren B.
      • Gingeras T.
      • Gilbert D.M.
      • Groudine M.
      • Bender M.
      • Kaul R.
      • Canfield T.
      • Giste E.
      • Johnson A.
      • Zhang M.
      • Balasundaram G.
      • Byron R.
      • Roach V.
      • Sabo P.J.
      • Sandstrom R.
      • Stehling A.S.
      • Thurman R.E.
      • Weissman S.M.
      • Cayting P.
      • Hariharan M.
      • Lian J.
      • Cheng Y.
      • Landt S.G.
      • Ma Z.
      • Wold B.J.
      • Dekker J.
      • Crawford G.E.
      • Keller C.A.
      • Wu W.
      • Morrissey C.
      • Kumar S.A.
      • Mishra T.
      • Jain D.
      • Byrska-Bishop M.
      • Blankenberg D.
      • Lajoie B.R.
      • Jain G.
      • Sanyal A.
      • Chen K.B.
      • Denas O.
      • Taylor J.
      • Blobel G.A.
      • Weiss M.J.
      • Pimkin M.
      • Deng W.
      • Marinov G.K.
      • Williams B.A.
      • Fisher-Aylor K.I.
      • Desalvo G.
      • Kiralusha A.
      • Trout D.
      • Amrhein H.
      • Mortazavi A.
      • Edsall L.
      • McCleary D.
      • Kuan S.
      • Shen Y.
      • Yue F.
      • Ye Z.
      • Davis C.A.
      • Zaleski C.
      • Jha S.
      • Xue C.
      • Dobin A.
      • Lin W.
      • Fastuca M.
      • Wang H.
      • Guigo R.
      • Djebali S.
      • Lagarde J.
      • Ryba T.
      • Sasaki T.
      • Malladi V.S.
      • Cline M.S.
      • Kirkup V.M.
      • Learned K.
      • Rosenbloom K.R.
      • Kent W.J.
      • Feingold E.A.
      • Good P.J.
      • Pazin M.
      • Lowdon R.F.
      • Adams L.B.
      An encyclopedia of mouse DNA elements (Mouse ENCODE).
      Figure thumbnail gr10
      Figure 10HNF4A-dominated enterocyte and HNF4A-absent ISC regulatory regions behave distinctly in response to microbes and HFM. (A) Organized heatmap of merged accessible chromatin peak calls for sites that were significantly enriched in at least 1 of the 4 sorted IEC cell types (red).
      • Raab J.R.
      • Tulasi D.Y.
      • Wager K.E.
      • Morowitz J.M.
      • Magness S.T.
      • Gracz A.D.
      Quantitative classification of chromatin dynamics reveals regulators of intestinal stem cell differentiation.
      Jejunal DHS sites
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      and H3K4me2 sites from goblet cells
      • Kim T.H.
      • Li F.
      • Ferreiro-Neira I.
      • Ho L.L.
      • Luyten A.
      • Nalapareddy K.
      • Long H.
      • Verzi M.
      • Shivdasani R.A.
      Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity.
      are also included. Groups of sites that are accessible in enterocytes and ISCs are further broken down into whether they are accessible in other (restricted) or only their IEC type (specific). A subset of sites are accessible across IEC subtypes (pan-accessible). Corresponding accessible chromatin data overlap with other mouse tissues (blue) identifies patterns of enrichment.
      • Camp J.G.
      • Frank C.L.
      • Lickwar C.R.
      • Guturu H.
      • Rube T.
      • Wenger A.M.
      • Chen J.
      • Bejerano G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.
      ,
      • Mouse E.C.
      • Stamatoyannopoulos J.A.
      • Snyder M.
      • Hardison R.
      • Ren B.
      • Gingeras T.
      • Gilbert D.M.
      • Groudine M.
      • Bender M.
      • Kaul R.
      • Canfield T.
      • Giste E.
      • Johnson A.
      • Zhang M.
      • Balasundaram G.
      • Byron R.
      • Roach V.
      • Sabo P.J.
      • Sandstrom R.
      • Stehling A.S.
      • Thurman R.E.
      • Weissman S.M.
      • Cayting P.
      • Hariharan M.
      • Lian J.
      • Cheng Y.
      • Landt S.G.
      • Ma Z.
      • Wold B.J.
      • Dekker J.
      • Crawford G.E.
      • Keller C.A.
      • Wu W.
      • Morrissey C.
      • Kumar S.A.
      • Mishra T.
      • Jain D.
      • Byrska-Bishop M.
      • Blankenberg D.
      • Lajoie B.R.
      • Jain G.
      • Sanyal A.
      • Chen K.B.
      • Denas O.
      • Taylor J.
      • Blobel G.A.
      • Weiss M.J.
      • Pimkin M.
      • Deng W.
      • Marinov G.K.
      • Williams B.A.
      • Fisher-Aylor K.I.
      • Desalvo G.
      • Kiralusha A.
      • Trout D.
      • Amrhein H.
      • Mortazavi A.
      • Edsall L.
      • McCleary D.
      • Kuan S.
      • Shen Y.
      • Yue F.
      • Ye Z.
      • Davis C.A.
      • Zaleski C.
      • Jha S.
      • Xue C.
      • Dobin A.
      • Lin W.
      • Fastuca M.
      • Wang H.
      • Guigo R.
      • Djebali S.
      • Lagarde J.
      • Ryba T.
      • Sasaki T.
      • Malladi V.S.
      • Cline M.S.
      • Kirkup V.M.
      • Learned K.
      • Rosenbloom K.R.
      • Kent W.J.
      • Feingold E.A.
      • Good P.J.
      • Pazin M.
      • Lowdon R.F.
      • Adams L.B.
      An encyclopedia of mouse DNA elements (Mouse ENCODE).
      Groups of sites that correspond to enterocyte sites are marked with a straight blue line, and ISC but not enterocytes sites are marked with straight red lines. (B) Heatmap for HNF4A binding (yellow) and computationally detected HNF4A motif (orange) for sites ordered as in (A). These data are summarized with descending moving means (500 site window, 1 site step). A similar pattern is seen using a previously published jejunum HNF4A ChIP-seq dataset (teal).
      • Verzi M.P.
      • Shin H.
      • San Roman A.K.
      • Liu X.S.
      • Shivdasani R.A.
      Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.
      Duodenum SMAD4 ChIP-seq dataset (brown).
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      (C) Heatmap for H3K27ac differential binding for sites ordered as in (A). These data are summarized with descending moving means (500 site window, 1 site step). (D) The log2FC for H3K27ac sites that are significantly different in at least one comparison for the CV+HFM/GF+HFM comparison grouped by their linked neighboring gene into 1 of 5 compartments based on preferential expression along the crypt-villus axis.
      • Moor A.E.
      • Harnik Y.
      • Ben-Moshe S.
      • Massasa E.E.
      • Rozenberg M.
      • Eilam R.
      • Bahar Halpern K.
      • Itzkovitz S.
      Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis.
      Within the compartments, H3K27ac sites are ordered by random. H3K27ac sites that are significant by the comparison on the Y-axis are red dots. All nonsignificant sites are blue. Yellow dashed lines are average for all sites within that compartment. (E) GREAT GO term enrichment for enterocyte-specific sites. (F) GREAT GO term enrichment for ISC-specific sites. (G) Motif enrichment at ATAC-seq sites linked to ISC-specific and Ent-specific groups versus CV+HFM and GF+HFM groups. For each group the reciprocal direction is used as the background (i.e., ISC-specific input versus Ent-specific background). The –log10 P values are plotted. Both directions are plotted on the same axis with each analysis separated by colored arrows. (H) Groups of accessible sites (red bars) further organized by if they are bound by HNF4A (yellow bars) show dependence on HNF4A binding for decreased enrichment in CV+HFM/GF+HFM H3K27ac signal at enterocyte-specific sites and increased (I) ISC-specific sites on average.
      We overlapped these accessible chromatin sites with HNF4A binding from our data and identified that up to 60% of the pan-accessible sites were also bound by HNF4A (Figure 10B). Enterocyte-specific sites rose to a substantial 80% overlap with HNF4A binding (Figure 10B). In contrast, sites without enterocyte accessibility, including the large group of ISC-specific and -restricted sites, had limited to no HNF4A binding (∼10%) in our IEC preparation (Figure 10B). We confirmed this pattern using a published dataset for HNF4A binding in mouse jejunal IECs (Figure 10B).
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      ,
      • San Roman A.K.
      • Aronson B.E.
      • Krasinski S.D.
      • Shivdasani R.A.
      • Verzi M.P.
      Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2.
      We computationally detected the HNF4A motif at 30% of the sites with enterocyte accessibility and only 10% of the sites without accessibility in enterocytes. This suggests that sites that are accessible in ISC, but not enterocytes, are not typically conventionally bound by HNF4A in any circumstance. SMAD Family Member 4 (SMAD4) has recently been shown to contribute to enterocyte differentiation with HNF4A.
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      Although we see substantial coincidence of SMAD4 and HNF4A binding, the SMAD4 binding proportion at enterocyte-specific sites is reduced (∼20%) as compared with pan-accessible sites (60%), suggesting HNF4A contributes more exclusively to enterocyte-specific sites (Figure 10B).
      Finally, to query the impact of microbial and nutritional signals on the different types of regulatory regions, we compared the relative H3K27ac signal for each site for our 4 comparisons (Figure 10C). Surprisingly, intestinal accessibility groups had substantially different H3K27ac responses for different comparisons. CV+HFM/CV and CV/GF show reduction of H3K27ac signal at enterocyte-specific sites. This reduction is even more substantial in CV+HFM/GF+HFM, with the GF+HFM/GF comparison increasing. We identified the opposite effect when looking at ISC-specific sites: substantial H3K27ac signal increases in CV/GF, CV+HFM/CV, and especially CV+HFM/GF+HFM, and reduced signal in GF+HFM/GF (Figure 10C). This difference implies a major impact of HFM, in the presence or absence of microbes, is manipulation of the related crypt-villus, ISC-enterocyte, or proliferation-differentiation axis (Figure 10D). We found that enterocyte-specific sites are enriched near genes involved in carbohydrate metabolism, FAO, and lipid catabolism including Abcd3, Acaa2, Acox1, Crot, Ppargc1a, Acsl1, and Ppara (Figure 10E and F). HNF4A and other nuclear receptor motifs that were enriched in GF+HFM regulatory regions are also enriched in these enterocyte-specific sites (Figure 10G). Although this property may be attributable to enterocyte-specific regions behaving as a group or reminiscent of a change in the proportional number of a given cell type, we surprisingly found that only enterocyte-specific sites also bound by HNF4A showed altered H3K27ac changes on average (Figure 10H and I). HNF4A may therefore have a substantial influence on interacting microbial and nutritional enterocyte-specific regulatory regions.

      Discussion

      Physiologic responses by the intestine to microbiota and diet must be understood as dynamic processes. The postprandial response typically takes 8 hours for lipid absorption in mice
      • Uchida A.
      • Whitsitt M.C.
      • Eustaquio T.
      • Slipchenko M.N.
      • Leary J.F.
      • Cheng J.X.
      • Buhman K.K.
      Reduced triglyceride secretion in response to an acute dietary fat challenge in obese compared to lean mice.
      and can be influenced by host history, including diet, microbes, and circadian rhythm.
      • Martinez-Guryn K.
      • Hubert N.
      • Frazier K.
      • Urlass S.
      • Musch M.W.
      • Ojeda P.
      • Pierre J.F.
      • Miyoshi J.
      • Sontag T.J.
      • Cham C.M.
      • Reardon C.A.
      • Leone V.
      • Chang E.B.
      Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids.
      ,
      • Wang Y.
      • Kuang Z.
      • Yu X.
      • Ruhn K.A.
      • Kubo M.
      • Hooper L.V.
      The intestinal microbiota regulates body composition through NFIL3 and the circadian clock.
      ,
      • Mukherji A.
      • Kobiita A.
      • Ye T.
      • Chambon P.
      Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs.
      ,
      • Uchida A.
      • Whitsitt M.C.
      • Eustaquio T.
      • Slipchenko M.N.
      • Leary J.F.
      • Cheng J.X.
      • Buhman K.K.
      Reduced triglyceride secretion in response to an acute dietary fat challenge in obese compared to lean mice.
      Colonization of GF mice leads to transcriptional responses that remain dynamic for weeks and are dependent on the composition of the microbial community.
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
      • van Baarlen P.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      ,
      • El Aidy S.
      • van Baarlen P.
      • Derrien M.
      • Lindenbergh-Kortleve D.J.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Samsom J.N.
      • Nieuwenhuis E.E.
      • Kleerebezem M.
      Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice.
      The intestinal epithelial layer turns over every 3–5 days, with the potential to alter IEC subtypes and crypt-villus architecture while simultaneously making acute transcriptional changes to existing IECs on the order of minutes.
      • Heppert J.K.
      • Davison J.M.
      • Kelly C.
      • Mercado G.P.
      • Lickwar C.R.
      • Rawls J.F.
      Transcriptional programmes underlying cellular identity and microbial responsiveness in the intestinal epithelium.
      ,
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
      • van Baarlen P.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      ,
      • Zeituni E.M.
      • Wilson M.H.
      • Zheng X.
      • Iglesias P.A.
      • Sepanski M.A.
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      • Zheng Y.
      • Farber S.A.
      Endoplasmic reticulum lipid flux influences enterocyte nuclear morphology and lipid-dependent transcriptional responses.
      ,
      • Cheng H.
      • Leblond C.P.
      Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine: V—unitarian theory of the origin of the four epithelial cell types.
      ,
      • Al-Dewachi H.S.
      • Wright N.A.
      • Appleton D.R.
      • Watson A.J.
      Cell population kinetics in the mouse jejunal crypt.
      ,
      • Haber A.L.
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      A single-cell survey of the small intestinal epithelium.
      • Shamir M.
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      • Milo R.
      SnapShot: timescales in cell biology.
      • Mucunguzi O.
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      Identification of the principal transcriptional regulators for low-fat and high-fat meal responsive genes in small intestine.
      • Nyima T.
      • Muller M.
      • Hooiveld G.J.
      • Morine M.J.
      • Scotti M.
      Nonlinear transcriptomic response to dietary fat intake in the small intestine of C57BL/6J mice.
      • Xie Y.
      • Ding F.
      • Di W.
      • Lv Y.
      • Xia F.
      • Sheng Y.
      • Yu J.
      • Ding G.
      Impact of a highfat diet on intestinal stem cells and epithelial barrier function in middleaged female mice.
      • Mah A.T.
      • Van Landeghem L.
      • Gavin H.E.
      • Magness S.T.
      • Lund P.K.
      Impact of diet-induced obesity on intestinal stem cells: hyperproliferation but impaired intrinsic function that requires insulin/IGF1.
      We aimed here to understand how the intestine integrates 2 distinct conditions simultaneously to coordinate functional genomic activity, and specifically how microbes modify the response to a HFM. Focusing on a single early stage in the postprandial process, we identified a response with substantial complexity but also with clear contributions from PPARA, proliferation pathways, and HNF4A-driven enterocyte differentiation.
      At the level of H3K27ac and RNA-seq, our results indicate that PPARA signaling is preferentially engaged after HFM in the absence of microbes. We speculate that the high level of Ppara and FAO gene transcription in GF and GF+HFM may contribute to reduced accumulation and absorption of dietary lipid as compared with CV after HFM and may have additional systemic consequences.
      • Martinez-Guryn K.
      • Hubert N.
      • Frazier K.
      • Urlass S.
      • Musch M.W.
      • Ojeda P.
      • Pierre J.F.
      • Miyoshi J.
      • Sontag T.J.
      • Cham C.M.
      • Reardon C.A.
      • Leone V.
      • Chang E.B.
      Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids.
      ,
      • Tazi A.
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      • Arena E.T.
      • Nigro G.
      • Pedron T.
      • Sansonetti P.J.
      Disentangling host-microbiota regulation of lipid secretion by enterocytes: insights from commensals Lactobacillus paracasei and Escherichia coli.
      ,
      • Araujo J.R.
      • Tazi A.
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      • Nigro G.
      • Licandro H.
      • Demignot S.
      • Sansonetti P.J.
      Fermentation products of commensal bacteria alter enterocyte lipid metabolism.
      Signaling by PPARA and other nuclear receptors has been associated with gene expression in GF that is suppressed by microbes including in IECs from the colon and ileum.
      • Martinez-Guryn K.
      • Hubert N.
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      • Urlass S.
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      • Ojeda P.
      • Pierre J.F.
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      • Cham C.M.
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      • Leone V.
      • Chang E.B.
      Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids.
      ,
      • Backhed F.
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      • Nagy A.
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      The gut microbiota as an environmental factor that regulates fat storage.
      ,
      • Tazi A.
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      • Mulet C.
      • Arena E.T.
      • Nigro G.
      • Pedron T.
      • Sansonetti P.J.
      Disentangling host-microbiota regulation of lipid secretion by enterocytes: insights from commensals Lactobacillus paracasei and Escherichia coli.
      • Araujo J.R.
      • Tazi A.
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      • Nigro G.
      • Licandro H.
      • Demignot S.
      • Sansonetti P.J.
      Fermentation products of commensal bacteria alter enterocyte lipid metabolism.
      • Camp J.G.
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      • Guturu H.
      • Rube T.
      • Wenger A.M.
      • Chen J.
      • Bejerano G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.
      ,
      • Mukherji A.
      • Kobiita A.
      • Ye T.
      • Chambon P.
      Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs.
      ,
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
      • van Baarlen P.
      • Hooiveld G.
      • Levenez F.
      • Dore J.
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      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      Microbes may generally impact PPARA signaling along multiple intestinal regions. However, activation of PPARA reduces lipid droplet levels specifically in jejunum, and jejunal microbes fed a high-fat diet are capable of increasing lipid absorption.
      • Martinez-Guryn K.
      • Hubert N.
      • Frazier K.
      • Urlass S.
      • Musch M.W.
      • Ojeda P.
      • Pierre J.F.
      • Miyoshi J.
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      • Cham C.M.
      • Reardon C.A.
      • Leone V.
      • Chang E.B.
      Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids.
      ,
      • Karimian Azari E.
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      Possible role of intestinal fatty acid oxidation in the eating-inhibitory effect of the PPAR-alpha agonist Wy-14643 in high-fat diet fed rats.
      ,
      • Poteres E.
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      Selective regional alteration of the gut microbiota by diet and antibiotics.
      Therefore, both host and microbe regional-specific differences may contribute to lipid metabolism responses. Although we measured aspects of the host response to HFM at one early postprandial time point, additional research could uncover the impact of acute HFM on microbiota and how these dynamics change across the prandial cycle and in the context of chronic high-fat diet consumption.
      Our results identify FAO as a key process interactively regulated by microbes and HFM, but molecular mechanisms remain unresolved. We were interested to find that the same PPARA-associated genes that are induced by HFM are also induced by fasting (Figure 6K and L).
      • van den Bosch H.M.
      • Bunger M.
      • de Groot P.J.
      • van der Meijde J.
      • Hooiveld G.J.
      • Muller M.
      Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting.
      Fasting liberates stored fat to produce PPAR-activating FAs that can also be consumed by FAO to produce adenosine triphosphate.
      • Kersten S.
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      Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.
      The egg yolk meal used here is rich in FAs, which could act as PPARA ligands.
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      • Craney D.J.
      Lipidomics of the chicken egg yolk: high-resolution mass spectrometric characterization of nutritional lipid families.
      GF mice may be metabolically primed to initiate FAO to use a HFM as compared with CV mice.
      • Araujo J.R.
      • Tazi A.
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      • Nigro G.
      • Licandro H.
      • Demignot S.
      • Sansonetti P.J.
      Fermentation products of commensal bacteria alter enterocyte lipid metabolism.
      ,
      • Mukherji A.
      • Kobiita A.
      • Ye T.
      • Chambon P.
      Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs.
      ,
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
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      • Dore J.
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      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      Intestinal FAO and lipid storage might also be influenced by microbial metabolites such as δ-valerobetaine through the inhibition of carnitine synthesis,
      • Zhao M.
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      • Zheng L.
      TMAVA, a metabolite of intestinal microbes, is increased in plasma from patients with liver steatosis, inhibits gamma-butyrobetaine hydroxylase, and exacerbates fatty liver in mice.
      ,
      • Liu K.H.
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      Microbial metabolite delta-valerobetaine is a diet-dependent obesogen.
      acetate through the AMPK/PGC-1α/PPARα signaling pathway, or by c-Jun/NCoR regulation.
      • Backhed F.
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      Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.
      ,
      • Araujo J.R.
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      • Nigro G.
      • Licandro H.
      • Demignot S.
      • Sansonetti P.J.
      Fermentation products of commensal bacteria alter enterocyte lipid metabolism.
      ,
      • Mukherji A.
      • Kobiita A.
      • Ye T.
      • Chambon P.
      Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs.
      We were also interested to see that pharmacologic activation of PPARA not only led to activation of FAO genes that are typically suppressed by microbiota but also suppressed genes normally induced by microbiota such as Reg3g and Saa1 (Figure 6I).
      • Bunger M.
      • van den Bosch H.M.
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      • Kersten S.
      • Hooiveld G.J.
      • Muller M.
      Genome-wide analysis of PPARalpha activation in murine small intestine.
      This raises the intriguing possibility that PPARA signaling directly opposes proinflammatory programs in the small intestine.
      • Mukherji A.
      • Kobiita A.
      • Ye T.
      • Chambon P.
      Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs.
      In contrast to PPARA signaling, ISC and proliferation components were preferentially engaged by HFM in the presence of microbes. Increased IEC proliferation has been associated with microbiota colonization in mouse and zebrafish.
      • El Aidy S.
      • Merrifield C.A.
      • Derrien M.
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      • Hooiveld G.
      • Levenez F.
      • Dore J.
      • Dekker J.
      • Holmes E.
      • Claus S.P.
      • Reijngoud D.J.
      • Kleerebezem M.
      The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation.
      ,
      • Peck B.C.
      • Mah A.T.
      • Pitman W.A.
      • Ding S.
      • Lund P.K.
      • Sethupathy P.
      Functional transcriptomics in diverse intestinal epithelial cell types reveals robust microRNA sensitivity in intestinal stem cells to microbial status.
      ,
      • Rawls J.F.
      • Samuel B.S.
      • Gordon J.I.
      Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota.
      • Cheesman S.E.
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      Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88.
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      Promotion of intestinal epithelial cell turnover by commensal bacteria: role of short-chain fatty acids.
      Chronic high-fat diet is also associated with changes in crypt size and increased intestinal proliferation,
      • Beyaz S.
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      High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.
      ,
      • Xie Y.
      • Ding F.
      • Di W.
      • Lv Y.
      • Xia F.
      • Sheng Y.
      • Yu J.
      • Ding G.
      Impact of a highfat diet on intestinal stem cells and epithelial barrier function in middleaged female mice.
      ,
      • Mah A.T.
      • Van Landeghem L.
      • Gavin H.E.
      • Magness S.T.
      • Lund P.K.
      Impact of diet-induced obesity on intestinal stem cells: hyperproliferation but impaired intrinsic function that requires insulin/IGF1.
      ,
      • Mao J.
      • Hu X.
      • Xiao Y.
      • Yang C.
      • Ding Y.
      • Hou N.
      • Wang J.
      • Cheng H.
      • Zhang X.
      Overnutrition stimulates intestinal epithelium proliferation through beta-catenin signaling in obese mice.
      and in Drosophila, only in the presence of microbes does a high-fat diet induce proliferation in the intestine.
      • von Frieling J.
      • Faisal M.N.
      • Sporn F.
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      • Roeder T.
      A high-fat diet induces a microbiota-dependent increase in stem cell activity in the Drosophila intestine.
      We found that regulatory regions that have increased H3K27ac signal in response to both microbes and HFM (+HFM-up +CV-up) are associated with ISC regulatory regions and expression (Figure 6C and F). Interestingly, this ISC association emerged despite all our experiments here being from whole jejunal epithelium preparations that do not differentiate between ISC or enterocyte populations. On the basis of typical cell proliferation rates, we consider it unlikely that substantial changes in cell-type abundance occurred within the 2 hours after HFM.
      • Cheng H.
      • Leblond C.P.
      Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine: V—unitarian theory of the origin of the four epithelial cell types.
      ,
      • Al-Dewachi H.S.
      • Wright N.A.
      • Appleton D.R.
      • Watson A.J.
      Cell population kinetics in the mouse jejunal crypt.
      Our analysis of previously published data identifies that unlike CV, isolated GF jejunal ISCs appear to express genes associated with enterocytes relative to ISCs (Figure 9S).
      • Peck B.C.
      • Mah A.T.
      • Pitman W.A.
      • Ding S.
      • Lund P.K.
      • Sethupathy P.
      Functional transcriptomics in diverse intestinal epithelial cell types reveals robust microRNA sensitivity in intestinal stem cells to microbial status.
      Relationships between metabolism and proliferation and differentiation in the intestine are salient and complicated. Loss of Cpt1a expression in IECs leads to reduced FAO and proliferation,
      • Mihaylova M.M.
      • Cheng C.W.
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      • Sabatini D.M.
      • Yilmaz O.H.
      Fasting activates fatty acid oxidation to enhance intestinal stem cell function during homeostasis and aging.
      ,
      • Mana M.D.
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      • Yilmaz O.H.
      High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity.
      and loss of FAO in Hnf4agDKO or by chemical suppression is coincident with reduced ISC markers but increased transit-amplifying proliferation markers.
      • Chen L.
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      • Parthasarathy A.
      • Luo S.
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      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      In contrast, activation of FAO with a PPARA agonist results in suppression of the MYC proliferation program in the intestine.
      • Bunger M.
      • van den Bosch H.M.
      • van der Meijde J.
      • Kersten S.
      • Hooiveld G.J.
      • Muller M.
      Genome-wide analysis of PPARalpha activation in murine small intestine.
      It remains unclear whether these impacts of FAO regulation on ISCs and proliferation are direct or indirect. Indeed, PPAR signaling and FAO components are more highly expressed in enterocytes (Figure 9Q, S, and T). The microbial metabolite lactate has been shown to both inhibit FAO and activate intestinal ISCs, consistent with our findings and underscoring that distinct microbial signals can link altered lipid metabolism and intestinal proliferation.
      • Araujo J.R.
      • Tazi A.
      • Burlen-Defranoux O.
      • Vichier-Guerre S.
      • Nigro G.
      • Licandro H.
      • Demignot S.
      • Sansonetti P.J.
      Fermentation products of commensal bacteria alter enterocyte lipid metabolism.
      ,
      • Lee Y.S.
      • Kim T.Y.
      • Kim Y.
      • Lee S.H.
      • Kim S.
      • Kang S.W.
      • Yang J.Y.
      • Baek I.J.
      • Sung Y.H.
      • Park Y.Y.
      • Hwang S.W.
      • O E
      • Kim K.S.
      • Liu S.
      • Kamada N.
      • Gao N.
      • Kweon M.N.
      Microbiota-derived lactate accelerates intestinal stem-cell-mediated epithelial development.
      HNF4A may sit at the intersection of microbial and nutritional signals and to intestinal adaptation generally.
      • Dubois V.
      • Staels B.
      • Lefebvre P.
      • Verzi M.P.
      • Eeckhoute J.
      Control of cell identity by the nuclear receptor HNF4 in organ pathophysiology.
      Here we identify a conserved correlation between microbially regulated genes and HNF4A-regulated genes in mouse jejunum (Figure 9K–M).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      We propose that the correlation is driven by the related function of HNF4A promoting enterocyte differentiation relative to proliferation and microbial colonization promoting proliferation relative to differentiation. In addition, the potential for Hnf4a loss to eventually lead to intestinal inflammation may compound similarities to microbial responsive genes.
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Darsigny M.
      • Babeu J.P.
      • Dupuis A.A.
      • Furth E.E.
      • Seidman E.G.
      • Levy E.
      • Verdu E.F.
      • Gendron F.P.
      • Boudreau F.
      Loss of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic inflammation resembling inflammatory bowel disease in mice.
      HNF4G, which also contributes to enterocyte differentiation and binds at the same sites as HNF4A in IECs, is transcriptionally microbially suppressed in the CV+HFM/GF+HFM comparison (Supplementary Table 2).
      • Davison J.M.
      • Lickwar C.R.
      • Song L.
      • Breton G.
      • Crawford G.E.
      • Rawls J.F.
      Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor hepatocyte nuclear factor 4 alpha.
      ,
      • Chen L.
      • Toke N.H.
      • Luo S.
      • Vasoya R.P.
      • Fullem R.L.
      • Parthasarathy A.
      • Perekatt A.O.
      • Verzi M.P.
      A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity.
      ,
      • Raab J.R.
      • Tulasi D.Y.
      • Wager K.E.
      • Morowitz J.M.
      • Magness S.T.
      • Gracz A.D.
      Quantitative classification of chromatin dynamics reveals regulators of intestinal stem cell differentiation.
      ,
      • Lindeboom R.G.
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      • Burgering B.M.
      • Snippert H.J.
      • Vermeulen M.
      Integrative multi-omics analysis of intestinal organoid differentiation.
      HNF4A is proposed to function primarily as an activator genome-wide in IECs, although it can also function as a repressor and is expressed in both enterocytes and ISCs.
      • Chen L.
      • Vasoya R.P.
      • Toke N.H.
      • Parthasarathy A.
      • Luo S.
      • Chiles E.
      • Flores J.
      • Gao N.
      • Bonder E.M.
      • Su X.
      • Verzi M.P.
      HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice.
      ,
      • Santangelo L.
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      • Amicone L.
      • Tripodi M.
      The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4alpha.
      • Qu M.
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      • Hirota T.
      • Kay S.A.
      Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks.
      • Rodriguez J.C.
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      The hepatocyte nuclear factor 4 (HNF-4) represses the mitochondrial HMG-CoA synthase gene.
      • Martinez-Jimenez C.P.
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      Methods

      Mouse Husbandry

      All mice used in this study were in the C57BL/6J strain originally sourced from Jackson Laboratories (Bar Harbor, ME) and maintained in the National Gnotobiotic Rodent Resource Center at the University of North Carolina (UNC) at Chapel Hill. Male mice were reared under GF conditions or reared GF and colonized for 14 days with a conventional microbiota from feces of C57BL/6J SPF mice (CV). Mouse colonization was performed exactly as previously described.
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      Whereas conventionalization of GF mice stimulates temporally dynamic host responses in the intestine,
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      Normalization of host intestinal mucus layers requires long-term microbial colonization.
      we elected to focus on 14 days after colonization for our endpoint because it is sufficient for multiple rounds of epithelial renewal and for the initiation of a robust functional genomic host response.
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