Dynamic RNA polymerase II occupancy drives differentiation of the intestine under the direction of HNF4.

Terminal differentiation requires massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4,000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identify dynamic occupancy of RNA polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II occupancy and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, chromatin immunoprecipitation sequencing (ChIP-seq), and immunoprecipitation (IP) mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II occupancy at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms that drive differentiation gene expression and find that pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in renewing adult tissue.

A MTA2-SATB2 chromatin complex restrains colonic plasticity toward small intestine by retaining HNF4A at colonic chromatin.

Plasticity among cell lineages is a fundamental, but poorly understood, property of regenerative tissues. In the gut tube, the small intestine absorbs nutrients, whereas the colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screening, we identify MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrains colonic plasticity. MTA2 loss in the adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-occupies DNA with HNF4A, an activating pan-intestinal transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic chromatin, and accumulation on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through an HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages, and their release unleashes plasticity.

Stress-induced mucin 13 reductions drive intestinal microbiome shifts and despair behaviors.

Depression is a prevalent psychological condition with limited treatment options. While its etiology is multifactorial, both chronic stress and changes in microbiome composition are associated with disease pathology. Stress is known to induce microbiome dysbiosis, defined here as a change in microbial composition associated with a pathological condition. This state of dysbiosis is known to feedback on depressive symptoms. While studies have demonstrated that targeted restoration of the microbiome can alleviate depressive-like symptoms in mice, translating these findings to human patients has proven challenging due to the complexity of the human microbiome. As such, there is an urgent need to identify factors upstream of microbial dysbiosis. Here we investigate the role of mucin 13 as an upstream mediator of microbiome composition changes in the context of stress. Using a model of chronic stress, we show that the glycocalyx protein, mucin 13, is selectively reduced after psychological stress exposure. We further demonstrate that the reduction of Muc13 is mediated by the Hnf4 transcription factor family. Finally, we determine that deleting Muc13 is sufficient to drive microbiome shifts and despair behaviors. These findings shed light on the mechanisms behind stress-induced microbial changes and reveal a novel regulator of mucin 13 expression.

Smad4 Loss in the Mouse Intestinal Epithelium Alleviates the Pathological Fibrotic Response to Injury in the Colon.

Mucosal healing is associated with better clinical outcomes in patients with inflammatory bowel diseases (IBDs). Unresolved injury and inflammation, on the other hand, increases pathological fibrosis and the predisposition to cancer. Loss of Smad4, a tumor suppressor, is known to increase colitis-associated cancer in mouse models of chronic IBD. Since common biological processes are involved in both injury repair and tumor growth, we sought to investigate the effect of Smad4 loss on the response to epithelial injury. To this end, Smad4 was knocked out specifically in the intestinal epithelium and transcriptomic and morphological changes compared between wild type mice and Smad4 knock out mice after DSS-induced injury. We find that Smad4 loss alleviates pathological fibrosis and enhances mucosal repair. The transcriptomic changes specific to epithelium indicate molecular changes that affect epithelial extracellular matrix (ECM) and promote enhanced mucosal repair. These findings suggest that the biological processes that promote wound healing alleviate the pathological fibrotic response to DSS. Therefore, these mucosal repair processes could be exploited to develop therapies that promote normal wound healing and prevent fibrosis. NEW AND NOTEWORTHY: We show that transcriptomic changes due to Smad4 loss in the colonic epithelium alleviates the pathological fibrotic response to DSS in an IBD mouse model of acute inflammation. Most notably, we find that collagen deposition in the epithelial ECM, as opposed to that in the lamina propria, correlates with epithelial changes that enhance wound healing. This is the first report on a mouse model providing alleviated fibrotic response in a DSS-IBD mouse model in vivo .

Structural modification of C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives as potent inhibitors of the Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE signaling pathway is an attractive therapeutic target for the prevention and treatment of oxidative stress-associated diseases by activating the cellular expression of cytoprotective enzymes and proteins. Small molecule inhibitors can directly disrupt the Keap1-Nrf2 protein-protein interaction (PPI), resulting in elevated levels of Nrf2 protein and subsequent stimulation of related antioxidant responses. Previously, we found that 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives with an ether type C2-substituent on the benzene or naphthalene core exhibited potent inhibitory activities with IC50's in the submicromolar or nanomolar range. We here describe a more detailed structure-activity relationship study around the C2 substituents containing various polar linkers shedding new insight on their binding interactions with the Keap1 Kelch domain. The key observation from our findings is that the substituents at the C2-position of the benzene or naphthalene scaffold impact their inhibitory potencies in biochemical assays as well as activities in cell culture. The biochemical FP and TR-FRET assays revealed that the naphthalene derivatives 17b and 18 with an additional carboxylate at the C2 were the most active inhibitors against Keap1-Nrf2 PPI. In the cell-based assay, the two compounds were shown to be potent Nrf2 activators of the transcription of the Nrf2-dependent genes, such as HMOX2, GSTM3, and NQO1.

In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion.

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfß pathway.

Dynamic RNA Polymerase II Recruitment Drives Differentiation of the Intestine under the direction of HNF4.

Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identified dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of- function, ChIP-seq and IP mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms which drive differentiation gene expression and find pause-release of Pol II and post- transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health.

Multiple roles and regulatory mechanisms of the transcription factor HNF4 in the intestine.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

Hnf4 activates mimetic-cell enhancers to recapitulate gut and liver development within the thymus.

Mimetic cells are medullary thymic epithelial cells (mTECs) that mimic extra-thymic cell types to tolerize T cells to self-antigens. Here, we dissected the biology of entero-hepato mTECs, mimetic cells expressing gut- and liver-associated transcripts. Entero-hepato mTECs conserved their thymic identity yet accessed wide swaths of enterocyte chromatin and transcriptional programs via the transcription factors Hnf4α and Hnf4γ. Deletion of Hnf4α and Hnf4γ in TECs ablated entero-hepato mTECs and downregulated numerous gut- and liver-associated transcripts, with a primary contribution from Hnf4γ. Loss of Hnf4 impaired enhancer activation and CTCF redistribution in mTECs but did not impact Polycomb-mediated repression or promoter-proximal histone marks. By single-cell RNA sequencing, Hnf4 loss produced three distinct effects on mimetic cell state, fate, and accumulation. Serendipitously, a requirement for Hnf4 in microfold mTECs was discovered, which exposed a requirement for Hnf4γ in gut microfold cells and the IgA response. Study of Hnf4 in entero-hepato mTECs thus revealed mechanisms of gene control in the thymus and periphery alike.

RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells.

RAB11 small GTPases and associated recycling endosome have been localized to mitotic spindles and implicated in regulating mitosis. However, the physiological significance of such regulation has not been observed in mammalian tissues. We have used newly engineered mouse models to investigate intestinal epithelial renewal in the absence of single or double isoforms of RAB11 family members: Rab11a and Rab11b. Comparing with single knockouts, mice with compound ablation demonstrate a defective cell cycle entry and robust mitotic arrest followed by apoptosis, leading to a total penetrance of lethality within 3 days of gene ablation. Upon Rab11 deletion ex vivo, enteroids show abnormal mitotic spindle formation and cell death. Untargeted proteomic profiling of Rab11a and Rab11b immunoprecipitates has uncovered a shared interactome containing mitotic spindle microtubule regulators. Disrupting Rab11 alters kinesin motor KIF11 function and impairs bipolar spindle formation and cell division. These data demonstrate that RAB11A and RAB11B redundantly control mitotic spindle function and intestinal progenitor cell division, a mechanism that may be utilized to govern the homeostasis and renewal of other mammalian tissues.

ß-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that ß-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of ß-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant ß-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

Optimization of the C2 substituents on the 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acid scaffold for better inhibition of Keap1-Nrf2 protein-protein interaction.

Direct inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) reduces the ubiquitination and subsequent degradation of Nrf2, leading to Nrf2 accumulation in the cytosol and the nuclear translocation of Nrf2. Once inside the nucleus, Nrf2 binds to and activates the expression of antioxidant response element (ARE) genes involved in redox homeostasis and detoxification. Herein, we report a series of 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acid analogs with varying C2 substituents to explore the structure-activity relationships at this position of the central naphthalene core. The Keap1-binding activities were first screened with a fluorescence polarization (FP) assay followed by further evaluation of the more potent compounds using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay. It was found that compound 24a with C2-phthalimidopropyl group was the most potent in this series showing an IC50 of 2.5 nM in the TR-FRET assay with a Ki value in the subnanomolar range. Our docking study indicated that the C2-phthalimidopropyl group in compound 24a provided an extra hydrogen bonding interaction with the key residue Arg415 that may be responsible for the observed boost in binding affinity. In addition, compounds 12b, 15, and 24a were shown to activate the Nrf2 signaling pathway in NCM460D cells resulting in elevated mRNA levels of GSTM3, HMOX1 and NQO1 by 2.4-11.7 fold at 100 µM as compared to the vehicle control.

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

TGFB1 Induces Fetal Reprogramming and Enhances Intestinal Regeneration.

The adult gut epithelium has a remarkable ability to recover from damage. To achieve cellular therapies aimed at restoring and/or replacing defective gastrointestinal tissue, it is important to understand the natural mechanisms of tissue regeneration. We employed a combination of high throughput sequencing approaches, mouse genetic models, and murine and human organoid models, and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. Depletion of macrophages or genetic disruption of TGFB-signaling significantly impaired the regenerative response following irradiation. Murine intestinal regeneration is also characterized by a process where a fetal transcriptional signature is induced during repair. In organoid culture, TGFB1-treatment was necessary and sufficient to induce a transcriptomic shift to the fetal-like/regenerative state. The regenerative response was enhanced by the function of mesenchymal cells, which are also primed for regeneration by TGFB1. Mechanistically, integration of ATAC-seq, scRNA-seq, and ChIP-seq suggest that a regenerative YAP-SOX9 transcriptional circuit is activated in epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for the application of the TGFB-induced regenerative circuit in cellular therapy.

Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Moringa isothiocyanate-1 inhibits LPS-induced inflammation in mouse myoblasts and skeletal muscle.

This study aims to investigate the anti-inflammatory effects of moringa isothiocyanate-1 (MIC-1) extracted from seeds of Moringa oleifera Lam. in lipopolysaccharide (LPS)-induced inflammation models. MIC-1 decreased nitric oxide production and reduced the expression of pro-inflammatory markers (TNF-α, Ifn-α, IL-1ß, IL-6) in C2C12 myoblasts. The daily oral treatment of MIC-1 (80 mg/kg) for three days significantly reduced the expression of pro-inflammatory markers in gastrocnemius muscle tissue of LPS-treated C57BL/6 male mice. Transcriptomic analysis provided further insights into the inhibitory effects of MIC-1 on the LPS-induced inflammation, which suggested that MIC-1 affects inflammation and immunity-related genes in myoblasts and skeletal muscle tissue. MIC-1 inhibited the nuclear accumulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-treated myoblasts. Our data support the hypothesis that the MIC-1's effects in the muscle cells are mediated through the inhibition of the NF-κB translocation in the nucleus, which, in turn, results in immunomodulating and anti-inflammatory responses at the gene expression levels.

Genomic analysis of 1,25-dihydroxyvitamin D3 action in mouse intestine reveals compartment and segment-specific gene regulatory effects.

1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VD-induced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt (162 up, 210 down), villi (199 up, 63 down), or colon (102 up, 28 down), but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor-binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone.