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. 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. Vitamin D is an important nutrient with critical regulatory actions on intestinal physiology and function (1Christakos S. Vitamin D: a critical regulator of intestinal physiology.JBMR Plus. 2021; 5e10554Crossref Scopus (3) Google Scholar). Vitamin D is metabolized to become 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a hormone that activates the vitamin D receptor (VDR) to mediate the transcription of target genes. VDR-mediated gene transcription is a multistep process that involves VDR binding to target genes at both active promoters and distal regulatory elements as well as recruitment of coregulatory proteins (2Pike J.W. Christakos S. Biology and mechanisms of action of the vitamin D hormone.Endocrinol. Metab. Clin. North Am. 2017; 46: 815-843Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Although 1,25(OH)2D3 has been reported to regulate physiological processes in many tissues, the highest levels of VDR are present in the intestine, the major 1,25(OH)2D3 target tissue (3Lee S.M. Bishop K.A. Goellner J.J. O'Brien C.A. Pike J.W. Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.Endocrinology. 2014; 155: 2064-2076Crossref PubMed Scopus (26) Google Scholar, 4Cartwright J.A. Gow A.G. Milne E. Drummond D. Smith S. Handel I. et al.Vitamin D receptor expression in dogs.J. Vet. Intern. Med. 2018; 32: 764-774Crossref PubMed Scopus (13) Google Scholar). Studies in VDR null mice showed that deletion of VDR causes the loss of active calcium absorption in the proximal intestine, leading to hypocalcemia and rickets (5Van Cromphaut S.J. Dewerchin M. Hoenderop J.G. Stockmans I. Van Herck E. Kato S. et al.Duodenal calcium absorption in vitamin D receptor-knockout mice: Functional and molecular aspects.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13324-13329Crossref PubMed Scopus (461) Google Scholar). In addition, intestine-specific transgenic expression of VDR in VDR null mice normalized calcium absorption, serum calcium, and prevented the development of rickets (6Xue Y.B. Fleet J.C. Intestinal vitamin D receptor is required for normal calcium and bone metabolism in mice.Gastroenterology. 2009; 136: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Dhawan P. Veldurthy V. Yehia G. Hsaio C. Porta A. Kim K.I. et al.Transgenic expression of the vitamin D receptor restricted to the ileum, cecum, and colon of vitamin D receptor knockout mice rescues vitamin D receptor-dependent rickets.Endocrinology. 2017; 158: 3792-3804Crossref PubMed Scopus (19) Google Scholar). These findings indicate that a primary role of VDR and 1,25(OH)2D3 signaling during growth is the regulation of intestinal calcium absorption needed for calcium homeostasis and bone mineralization. Although most studies have focused on the duodenum, our recent studies have shown that the distal segments of the intestine also play an important role in VDR-mediated intestinal calcium absorption and bone mineralization (7Dhawan P. Veldurthy V. Yehia G. Hsaio C. Porta A. Kim K.I. et al.Transgenic expression of the vitamin D receptor restricted to the ileum, cecum, and colon of vitamin D receptor knockout mice rescues vitamin D receptor-dependent rickets.Endocrinology. 2017; 158: 3792-3804Crossref PubMed Scopus (19) Google Scholar, 8Li S. De La Cruz J. Hutchens S. Mukhopadhyay S. Criss Z.K. Aita R. et al.Analysis of 1,25-dihydroxyvitamin D3 genomic action reveals calcium-regulating and calcium-independent effects in mouse intestine and human enteroids.Mol. Cell Biol. 2020; 41e00372-20Crossref PubMed Scopus (11) Google Scholar, 9Jiang H. Horst R.L. Koszewski N.J. Goff J.P. Christakos S. Fleet J.C. Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides.J. Steroid Biochem. Mol. Biol. 2020; 198105574Crossref PubMed Scopus (6) Google Scholar). In addition to maintenance of calcium homeostasis, many other beneficial intestinal effects of 1,25(OH)2D3 have been described including anti-inflammatory effects, maintenance of intestinal barrier function, and protection against colitis and colon cancer, suggesting the existence of multiple, diverse 1,25(OH)2D3 functions across the length of the intestine (1Christakos S. Vitamin D: a critical regulator of intestinal physiology.JBMR Plus. 2021; 5e10554Crossref Scopus (3) Google Scholar). In addition to functional differences that exist along the proximal-to-distal axis, the proximal segments of the intestine have epithelial cells organized along a crypt–villus axis. Several studies have shown that 1,25(OH)2D3 action varies along the crypt–villus axis. In duodenal mid-villus cells, 1,25(OH)2D3 rapidly stimulates calcium extrusion (10Walters J.R. Weiser M.M. Calcium transport by rat duodenal villus and crypt basolateral membranes.Am. J. Physiol. 1987; 252: G170-G177PubMed Google Scholar) but slower effects of 1,25(OH)2D3 on crypt cells program the intestine for improved calcium absorption as the cells differentiate and migrate into the villus (11Wu J.C. Smith M.W. Lawson D.E. Time dependency of 1,25(OH)2D3 induction of calbindin mRNA and calbindin expression in chick enterocytes during their differentiation along the crypt-villus axis.Differentiation. 1992; 51: 195-200Crossref PubMed Scopus (11) Google Scholar). Meanwhile, VDR loss increases colon epithelial cell proliferation and alters the contribution of Lgr5+ stem cells to the maintenance of the intestinal epithelium (12DeLuca H.F. Franceschi R.T. Halloran B.P. Massaro E.R. Molecular events involved in 1,25-dihydroxyvitamin D3 stimulation of intestinal calcium transport.Fed. Proc. 1982; 41: 66-71PubMed Google Scholar, 13Peregrina K. Houston M. Daroqui C. Dhima E. Sellers R.S. Augenlicht L.H. Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.Carcinogenesis. 2015; 36: 25-31Crossref PubMed Scopus (38) Google Scholar). In spite of the recognized regulatory role of 1,25(OH)2D3–VDR signaling in intestinal biology, the mechanisms involved in VDR-mediated regulation of these diverse functions remain incomplete and genomic studies of 1,25(OH)2D3 action in the intestine are sparse. In addition, the diversity and complexity of 1,25(OH)2D3 signaling with respect to proximal–distal and crypt–villus axes have not as yet been evaluated when considering intestinal 1,25(OH)2D3 action. In this study, we used a series of complementary genomic tools (i.e., RNA-seq, VDR ChIP-seq, ATAC-seq) to identify 1,25(OH)2D3-responsive target genes across the proximal-distal and small intestine (SI) crypt–villus axis. Our findings show that while a number of 1,25(OH)2D3-regulated genes are common across SI villus, SI crypt, and colon, the majority of 1,25(OH)2D3-regulated transcripts have compartment-restricted regulation patterns. Gene ontology (GO) and pathway analysis of the 1,25(OH)2D3-regulated transcripts from each compartment indicated regulation of unique biological functions, independent of calcium homeostasis, including regulation of RNA metabolic processes, tight junctions, metabolism of xenobiotics, lipid metabolic processes, and HIF1 signaling. However, not all VDR-regulated genes have VDR-binding peaks, suggesting that some transcript levels effects of 1,25(OH)2D3 are indirect and may be due in part to the contribution of other transcription factors. Our findings are the first to define 1,25(OH)2D3-molecular actions across the critical proximal–distal and crypt–villus axes that define the functional characteristics of the intestine and suggest novel mechanisms that may account for intestine compartment-specific functions of 1,25(OH)2D3. We confirmed the quality of our isolation of the various intestinal segments in two ways. First, we visually examined the small intestinal villus and crypt preparations to confirm that they were pure (see Fig. S1 for representative pictures of the isolated small intestinal crypts and villi). In addition, we examined our RNA-seq data to identify the transcript level differences across the three compartments. As expected, there were many differentially expressed genes between the small intestinal villus [5,114, 1% false detection rate (FDR), 2-fold change] or crypts (3554 differentially expressed gene (DEG)) and the colon. This included 135-fold higher mRNA levels of the SI marker lactase (Lct) in the villus versus colon and a 189-fold greater expression of the colon marker, carbonic anhydrase 1 (Car1) mRNA, in colon versus villus (Table S1). In the 3669 DEG we observed between the crypt and villus compartments, we observed that transcripts for markers of differentiated small intestinal epithelial cells were enriched in the SI villus (Lct, 3.49 up, S100g, 3.56 up, Trpv6, 3.71 up), while expression of the intestinal stem cell marker Lgr5 was elevated significantly in crypts (+12.2 fold). Collectively, these compartment-level differences in transcript levels confirm the quality of our isolation procedure. A summary of the impact of 1,25(OH)2 D treatment on intestinal gene expression is presented in Table S2. As expected, 1,25(OH)2D3 treatment significantly induced the expression of several genes known to be involved in intestinal calcium absorption; Trpv6, S100g, and Atp2b1 (Fig. 1). In addition, our analysis showed that Vdr mRNA levels were not dramatically different across segments nor were they strongly regulated by 1,25(OH)2 D treatment (Fig. 1B). Figure 2 shows that 968 transcripts were differentially regulated by 1,25(OH)2 D across the three compartments at the 5% FDR (A Venn Diagram showing the differentially expressed genes at 10% FDR is provided as Fig. S2.) Only 55 of these genes were common across all compartments (including the known 1,25(OH)2D3 target genes Cyp24a1, Trpv6, S100g, Slc30a10, and Atp2b1), while 78% of the 1,25(OH)2D3-regulated transcripts were specific to just one compartment. Ninety three percent of the 55 common targets were upregulated by 1,25(OH)2D3 treatment. Similarly, more than 80% of 1,25(OH)2D3-regulated genes in the SI villus and colon were induced. In contrast, only 56% of SI crypt transcripts were upregulated.Figure 2Venn diagram depicting significantly differentially expressed transcripts in the small intestine crypt, small intestine villus, or colon. Mice were treated with 1,25(OH)2D3 (10 ng/g, 4 h) and RNA from the three compartments was used for RNA-seq analysis. Differential expression was determined using DeSeq2 (5% FDR). The percentage of upregulated (UP) and downregulated transcripts (DN) are prevented for the total number of differentially expressed transcripts by tissue and for the compartment-specific transcripts. 1,25(OH)2D3, 1,25-dihydroxyvitamin D3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We compared our 1,25(OH)2D3-differentially expressed gene list with data on 1,25(OH)2D3-regulated transcripts in the SI that was previously published by Lee et al. (14Lee S.M. Riley E.M. Meyer M.B. Benkusky N.A. Plum L.A. DeLuca H.F. et al.1,25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes in the proximal intestine that is enriched for calcium-regulating components.J. Biol. Chem. 2015; 290: 18199-18215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Of the 719 DEG reported by them, 486 transcripts were identified in our intestinal RNA-seq data. One hundred seventy-nine of these matched to the 10% DEG list from at least one of the intestinal compartments (Table S2 and Fig. S3). Thirty eight transcripts were upregulated by 1,25(OH)2D3 in all three compartments and also in the Lee et al. dataset, including S100g, Trpv6, Atp2b1, Cyp24a1, and Slc30a10. We examined the 1,25(OH)2D3-regulated gene list from each compartment for enrichment of GO terms and pathways (Table 1 and Table S3 (Pathways), S4 (GO Up enrichment), and S5 (GO down enrichment)). Distinct functional categories of genes were identified for each compartment, including enrichment of GO terms for "lipid metabolic processes" and "ion transport" in villus, terms related to rRNA, RNA, and ncRNA processing in the crypts, and "Negative regulation of cell population proliferation" and "Regulation of Cell migration" in colon.Table 1Summary of GO enrichment for 1,25(OH)2D3-regulated genes by compartmentTissueEnrichmentTopicGenes in DEG listCryptUprRNA/RNA/ncRNA processing76Ribosome/cell component biogenesis103CryptDownResponse to chemical stimulus140Positive regulation of ion transport54VillusUpIon transport103Lipid metabolic processes72VillusDownPrimary metabolic processes78Cell activation22ColonUpNegative regulation of cell population proliferation39Regulation of cell migration43ColonDownCell developmental processes33Regulation of cellular component organization24 Open table in a new tab Using VDR ChIP-seq, we found many 1,25(OH)2D3-induced VDR-binding peaks in each intestinal compartment: 12,719 in SI crypt, 18,083 in SI villus, and 22,888 in colon. The ChIP-Seq signal was similar across compartments and VDR ChIP peaks averaged ∼ 1000 bp wide (Fig. 3A). Included in our VDR ChIP-seq peaks were the previously reported VDR-binding peaks in the Cyp24a1 gene (TSS at -0.2 kb and downstream enhancer peaks at +35, +37, +39, and +43 kb); the Trpv6 gene (at -2, -4 kb); and Slc30a10 gene (e.g. robust peaks at +29, +32, and +48 kb) (14Lee S.M. Riley E.M. Meyer M.B. Benkusky N.A. Plum L.A. DeLuca H.F. et al.1,25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes in the proximal intestine that is enriched for calcium-regulating components.J. Biol. Chem. 2015; 290: 18199-18215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). More than 60% of the VDR-binding peaks in the SI villus and crypt and 44% of the VDR-binding peaks in the colon coincided with the ATAC-Seq peaks from these same tissues (See Fig. S4). In contrast, some 1,25(OH)2D3-induced VDR peaks did not coincide with an ATAC-seq peak and this suggests that 1,25(OH)2D3 treatment revealed regulatory sites that were either silent under basal conditions or under the ATAC detection limit in untreated mouse intestine (e.g. Fig. S5 for the Slc30a10 gene). An evaluation of the VDR-binding peaks for known transcription factor binding site motifs revealed that the VDR-RXR DR3 motif was the most enriched motif in all three compartments (Fig. 3B and Table S6)). In addition, 16 other motifs for intestine-expressed transcription factors were enriched in the VDR ChIP-seq peaks (5% FDR, >1.5 fold enrichment over background DNA), including motifs for RAR, Bach1, HNF4a, JUN, and FOSL2 (in peaks from all three compartments); GATA4, YY1, MAFK (crypt and villus); HNF1, CEBP (crypt), and CDX2, TCF3, THRa (colon) (Fig. S6). We attributed the VDR-ChIP peaks to their nearest neighbor gene using GREAT in GSEA. This data was then used to identify the genes differentially expressed by 1,25(OH)2D3 that also had a VDR-binding peak associated with them (Fig. 3D). Approximately, 70% of the 1,25(OH)2D3-induced transcripts in SI villus and colon had a VDR-binding peak. In contrast, only 52% of 1,25(OH)2D3-induced crypt transcripts had VDR-binding peaks, while even fewer 1,25(OH)2D3-suppressed transcripts had them (villus 38.4%; colon 26.8%; SI crypt 21.1%). This suggests that the regulation of many genes, especially those induced in the crypt and suppressed in all compartments, were not direct 1,25(OH)2D3 target genes but may be a consequence of an upstream 1,25(OH)2D3-regulated event. One possibility for how 1,25(OH)2 D treatment could alter transcript levels independent of VDR binding to a gene regulatory region is that 1,25(OH)2D3 regulates the expression of other transcription factors that have 1,25(OH)2D3-independent downstream actions. Consistent with this hypothesis, bioinformatic analysis identified the protein class "transcription factors" as enriched in the 1,25(OH)2D3-regulated genes from crypt and colon (Table S7). In addition, we found that, of the transcription factors expressed in the colon (n = 307) or the SI (n = 348) of mice (15Zhou Q. Liu M. Xia X. Gong T. Feng J. Liu W. et al.A mouse tissue transcription factor atlas.Nat. Commun. 2017; 815089Crossref Scopus (45) Google Scholar), 50 were differentially expressed by 1,25(OH)2D3 treatment (16 induced and six suppressed transcription factor genes had a VDR binding site). Of the 50 1,25(OH)2D3-regulated transcription factor messages, 18 had enrichment of their downstream target genes in our dataset (Fig. 4 and Table S8). We next used Diffbind to compare VDR peaks across the three compartments to test whether compartment-specific regulation of genes by 1,25(OH)2 D treatment was due to differential binding of VDR to specific regulatory sites. Differential VDR binding was minimal between the small intestinal crypt and villus (80 crypt-enriched peaks, five villus-enriched peaks). In contrast, there were several hundred VDR peaks that were differentially enriched in either the colon or SI (Fig. 5A with images of the enriched peak profile in Fig. 5B). As shown in Figure 3, VDR peaks are more common for the induced genes so we evaluated the number of DEG with VDR-binding peaks that had both compartment-specific binding and induced expression. Our data show that fewer than 30% of the compartment-specific, differentially regulated transcripts also had compartment-specific differential VDR binding. This includes genes like Slc37a2, which has a VDR-binding site within an intronic enhancer in the SI that is absent in the colon, as well as Ptges, which has an VDR binding, intronic enhancer in colon that is lower in the SI (Fig. 5C). It is well established that 1,25(OH)2D3 is a critical regulator of intestinal physiology that controls calcium absorption (16Fleet J.C. The role of vitamin D in the endocrinology controlling calcium homeostasis.Mol. Cell Endocrinol. 2017; 453: 36-45Crossref PubMed Scopus (106) Google Scholar), enhances barrier function (17Fujita H. Sugimoto K. Inatomi S. Maeda T. Osanai M. Uchiyama Y. et al.Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes.Mol. Biol. Cell. 2008; 19: 1912-1921Crossref PubMed Scopus (314) Google Scholar, 18Kong J. Zhang Z. Musch M.W. Ning G. Sun J. Hart J. et al.Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier.Am. J. Physiol. Gastrointest Liver Physiol. 2008; 294: G208-G216Crossref PubMed Scopus (476) Google Scholar), regulates colonic inflammation (19Wang F. Johnson R.L. DeSmet M.L. Snyder P.W. Fairfax K.C. Fleet J.C. Vitamin D receptor-dependent signaling protects mice from dextran sulfate sodium-induced colitis.Endocrinology. 2017; 158: 1951-1963Crossref PubMed Scopus (20) Google Scholar), and suppresses colon cancer development (20Ferrer-Mayorga G. Larriba M.J. Crespo P. Munoz A. Mechanisms of action of vitamin D in colon cancer.J. Steroid Biochem. Mol. Biol. 2019; 185: 1-6Crossref PubMed Scopus (60) Google Scholar). Despite these diverse effects, the genomic mechanisms used by 1,25(OH)2D3 to regulate intestinal biology remain unknown. Previous studies have focused on intestinal effects of 1,25(OH)2D3 in the mature small intestinal enterocyte related primarily to calcium absorption or protection against barrier dysfunction (17Fujita H. Sugimoto K. Inatomi S. Maeda T. Osanai M. Uchiyama Y. et al.Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes.Mol. Biol. Cell. 2008; 19: 1912-1921Crossref PubMed Scopus (314) Google Scholar, 21Song Y. Peng X. Porta A. Takanaga H. Peng J.B. Hediger M.A. et al.Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice.Endocrinology. 2003; 144: 3885-3894Crossref PubMed Scopus (190) Google Scholar). In contrast, effects of 1,25(OH)2D3 in small intestinal crypts are only beginning to be defined and have been a matter of debate (10Walters J.R. Weiser M.M. Calcium transport by rat duodenal villus and crypt basolateral membranes.Am. J. Physiol. 1987; 252: G170-G177PubMed Google Scholar, 13Peregrina K. Houston M. Daroqui C. Dhima E. Sellers R.S. Augenlicht L.H. Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.Carcinogenesis. 2015; 36: 25-31Crossref PubMed Scopus (38) Google Scholar, 22Colston K.W. Mackay A.G. Finlayson C. Wu J.C. Maxwell J.D. Localisation of vitamin D receptor in normal human duodenum and in patients with coeliac disease.Gut. 1994; 35: 1219-1225Crossref PubMed Google Scholar). For example, it had been suggested that 1,25(OH)2D3 mediated transcription in intestinal villi but not in the crypts (23Reynolds C.J. Koszewski N.J. Horst R.L. Beitz D.C. Goff J.P. Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice.J. Steroid Biochem. Mol. Biol. 2019; 186: 56-60Crossref PubMed Scopus (7) Google Scholar). We attempted to resolve this issue by examining the molecular actions of 1,25(OH)2D3 across multiple functional compartments in the intestine, that is, the SI crypt, the SI villus, and the colonic epithelium. Consistent with our recent study (8Li S. De La Cruz J. Hutchens S. Mukhopadhyay S. Criss Z.K. Aita R. et al.Analysis of 1,25-dihydroxyvitamin D3 genomic action reveals calcium-regulating and calcium-independent effects in mouse intestine and human enteroids.Mol. Cell Biol. 2020; 41e00372-20Crossref PubMed Scopus (11) Google Scholar), 1,25(OH)2D3 treatment induced genes controlling intestinal Ca absorption in the all three compartments (i.e., Cyp24a1, Trpv6, S100g, and Atp2b1). However, only a small number of the 1,25(OH)2D3-regulated gene targets were common across the three compartments (5.7% of the total DEG). Instead, the majority of 1,25(OH)2D3-mediated genomic events were distinct and compartment specific (Fig. 2). Thus, our study reveals a complexity to intestinal 1,25(OH)2D3 action that had not previously been appreciated in genomic studies using SI mucosal scrapings (14Lee S.M. Riley E.M. Meyer M.B. Benkusky N.A. Plum L.A. DeLuca H.F. et al.1,25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes in the proximal intestine that is enriched for calcium-regulating components.J. Biol. Chem. 2015; 290: 18199-18215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) or cultured cells (24Wood R.J. Tchack L. Angelo G. Pratt R.E. Sonna L.A. DNA microarray analysis of vitamin D-induced gene expression in a human colon carcinoma cell line.Physiol. Genomics. 2004; 17: 122-129Crossref PubMed Scopus (62) Google Scholar, 25Costales-Carrera A. Fernandez-Barral A. Bustamante-Madrid P. Dominguez O. Guerra-Pastrian L. Cantero R. et al.Comparative study of organoids from patient-derived normal and tumor colon and rectal tissue.Cancers (Basel). 2020; 12: 2302Crossref Scopus (17) Google Scholar). A significant amount of research has been conducted to define the mechanisms controlling intestine-specific and intestine-segment–specific gene expression. This has defined transcription factors like CDX2, HNF4a/g, and GATA4/5/6 as central regulators of intestine cell identity (26Vierstra J. Rynes E. Sandstrom R. Zhang M. Canfield T. Hansen R.S. et al.Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.Science. 2014; 346: 1007-1012Crossref PubMed Scopus (167) Google Scholar) and GATA6, SATB2, and KLF4 as colon-enriched transcription factors (15Zhou Q. Liu M. Xia X. Gong T. Feng J. Liu W. et al.A mouse tissue transcription factor atlas.Nat. Commun. 2017; 815089Crossref Scopus (45) Google Scholar). However, few studies have explored how inducible gene expression is different across intestinal compartments. While there were only minor differences between VDR mRNA expression across the intestinal compartments, we found that ∼30% of compartment-specific 1,25(OH)2D3-induced transcripts could be explained by differential VDR binding to gene regulatory regions. Compartment-enriched VDR peaks were also found to differ in their enrichment of secondary transcription factor–binding motifs. This suggests that coordination between other transcription factors and VDR may contribute to differential binding and/or chromatin accessibility. For example, our findings of CDX2 and FOXA1 motifs at or near colon-enriched VDR-binding sites and HNF4a and GATA4 motifs at SI-enriched VDR-binding sites suggest that these transcription factors may promote or stabilize VDR binding at specific sites to mediate compartment-specific gene regulation. A stabilizing role would be similar to the role proposed for ETS1 in the regulation of 1,25(OH)2D3-mediated Cyp24a1 gene expression (27Dwivedi P.P. Omdahl J.L. Kola I. Hume D.K. May B.K. Regulation of rat cytochrome P450C24 (CYP24) gene expression - evidence for functional cooperation of Ras-activated Ets transcription factors with the vitamin D receptor in 1,25-dihydroxyvitamin D-3-mediated induction.J. Biol. Chem. 2000; 275: 47-55Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). However, these hypotheses must be formally tested. About 30% of the genes induced by 1,25(OH)2D3 in SI villus and colon and 48% of 1,25(OH)2D3-induced crypt transcripts did not have VDR-binding peaks. Also, between 60 to 79% of 1,25(OH)2D3-suppressed transcripts in each of the three compartments lacked a VDR-binding site. Thus, VDR binding does not predict mRNA expression. One hypothesis to explain VDR-independent and compartment-specific regulation of genes is that it is indirectly mediated through other transcription factors. In support of this hypothesis, we found 10 transcription factor genes that contained VDR-binding sites were differentially regulated by 1,25(OH)2 D treatment, and whose downstream targets were differentially regulated by 1,25(OH)2 D despite lacking VDR-binding sites near their genes. In the SI, this includes genes for transcription factors like NFATC3, which regulates intestinal differentiation (28Wang Q. Zhou Y. Weiss H.L. Chow C.W. Evers B.M. NFATc1 regulation of TRAIL expression in human intestinal cells.PLoS One. 2011; 6e19882Googl