The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a “non-specific” fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb(-/-) mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb-/- mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer. https://doi.org/10.7554/eLife.10903.001 eLife digest The digestive system in animals consists of a network of organs – including the liver, stomach, pancreas and intestines – that work together to break down food and deliver energy to the rest of the body. Many proteins called transcription factors help to guide the development of these organs and keep them healthy throughout life. Among these is a protein called HNF4α. In various diseases of the digestive system, such as gastric cancer or inflammatory bowel disease, the production of HNF4α is not properly regulated. Gene expression can be activated by transcription factors binding to regions of DNA called promoters. The gene that encodes HNF4α has two promoters called P1 and P2, and each produce several different versions of the HNF4α protein. The colon contains intestinal glands (also known as colonic crypts) that contain a lower part in which cells actively divide and an upper part of non-dividing cells that help with digestion. Previous studies have shown that if the mouse colon is unable to produce HNF4α, the structure of the crypts is disrupted. By studying crypts taken from the colon of mice, Chellappa et al. have now found that P1-HNF4α proteins are mainly produced at the top of the crypts, whereas P2-HNF4α proteins are found mainly at the bottom. Chellappa et al. then used two sets of genetically engineered mice: one that can only produce P1-HNFα proteins, and one that only has P2-HNFα proteins. Under normal conditions both sets of mice appeared healthy. However, differences became apparent if the mice were subjected to treatments that cause colitis or colitis-associated colon cancer. Mice that could only produce P1-HNF4α proteins were less susceptible to colitis and got fewer and smaller tumors than normal mice. By contrast, mice that could only produce P2-HNF4α experienced more colitis and developed more tumors than normal mice. Comparing the genes expressed in the colon cells of these two types of mice revealed several differences. In particular, much more of a pro-inflammatory protein called RELMβ was produced in P2-only mice. Chellappa et al. then proceeded to show that RELMβ is essential for the susceptibility of P2-mice to coliltis. Overall, the experiments show that P1-HNF4α and P2-HNF4α perform different tasks both in the healthy and the diseased mouse colon. In future it will be important to work out how the balance between the two sets of proteins is disrupted in diseases of the colon. https://doi.org/10.7554/eLife.10903.002 Introduction Hepatocyte nuclear factor 4alpha (HNF4α) (NR2Α1) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver, kidney, pancreas, stomach and intestine (Sladek et al., 1990). HNF4α is best known for its role in the liver where it is a master regulator of liver-specific gene expression and essential for adult and fetal liver function (Hayhurst et al., 2001; Kaestner, 2010; Bolotin et al., 2010; Odom, 2004). HNF4α is also known for its role in the pancreas where it regulates insulin secretion from beta cells (Gupta et al., 2007; 2005; Miura et al., 2006). Mutations in the HNF4Α gene and promoter regions are associated with Maturity Onset Diabetes of the Young 1 (MODY1) (Ellard and Colclough, 2006). In contrast, the role of HNF4α in the intestine has only recently been investigated. Knockout of the Hnf4a gene in the embryonic mouse colon results in disrupted crypt topology, and a decreased number of epithelial and mature goblet cells (Garrison et al., 2006), while the adult intestinal knockout shows defects in the balance between proliferation and differentiation as well as immune function, ion transport, epithelial barrier function and oxidative stress (Ahn et al., 2008; Cattin et al., 2009; Darsigny et al., 2009; 2010; Chahar et al., 2014). Dysregulation of the HNF4A gene is linked to several gastrointestinal disorders including colitis and colon cancer and a single nucleotide polymorphism in the HNF4A gene region is associated with ulcerative colitis (Ahn et al., 2008; Chellappa et al., 2012; Tanaka et al., 2006; Oshima et al., 2007; Barrett et al., 2009). While it is clear that HNF4α is critical for normal colon function, it is not known which transcript variant is the most relevant. There are two different promoters (proximal P1 and distal P2) in the HNF4α gene that are both active in the colon. The promoters are conserved from frog to human and, along with alternative splicing, give rise to nine different transcript variants of HNF4α (Huang et al., 2009) (Figure 1A). The major isoforms of the P1 promoter are HNF4α1/α2 while the P2 promoter gives rise to HNF4α7/α8: distinct first exons result in an altered A/B domain which harbors the activation function 1 (AF-1) while the DNA and ligand binding domains are identical. The two promoters are expressed under unique temporal and spatial conditions, with the large and small intestine being the only adult tissues that express both P1- and P2-HNF4α (Tanaka et al., 2006; Nakhei et al., 1998). While a loss of P1- but not P2-HNF4α has been noted in colon cancer (Chellappa et al., 2012; Tanaka et al., 2006), the specific roles of the HNF4α isoforms remain obscure. For example, P1-driven HNF4α acts as a tumor suppressor in mouse liver (Hatziapostolou et al., 2011; Walesky et al., 2013a). In contrast, the HNF4A gene and protein are amplified in human colon cancer (Cancer Genome Atlas Network, 2012; Zhang et al., 2014) although the different isoforms were not distinguished in those studies. We recently showed that ectopic expression of P1- but not P2-HNF4α decreased the tumorigenic potential of the human colon cancer cell line HCT116 in a mouse xenograft model (Vuong et al., 2015), suggesting that the different HNF4α isoforms may indeed play distinct roles in the colon. Here, we investigate the role of P1- and P2-HNF4α isoforms in the mouse colon using genetically engineered mice that express either the P1- or the P2-HNF4α isoforms (Briançon and Weiss, 2006). We show that in wildtype (WT) mice P1- and P2-HNF4α are expressed in different compartments in the colonic epithelium, interact with distinct sets of proteins, regulate the expression of unique sets of target genes, and play distinct roles during pathological conditions such as colitis and colitis-associated colon cancer (CAC). We also provide genetic and biochemical evidence indicating that RELMβ, a member of the RELM/FIZZ family of cytokines, plays a critical role in the response of HNF4α to colitis and appears to be both directly and indirectly regulated by HNF4α. Results Compartmentalization of P1- and P2-HNF4α in mouse colonic epithelium In the distal colon, the bottom two-thirds of the crypt and the top one-third, including surface epithelium, are functionally categorized as proliferative and differentiated compartments, respectively (Potten et al., 1997). We used monoclonal antibodies specific to the different HNF4α isoforms (Chellappa et al., 2012; Tanaka et al., 2006) (Figure 1A) to examine the distribution of P1- and P2-HNF4α along the crypt-surface axis. The P1/P2 antibody, which recognizes both P1- and P2-HNF4α, shows HNF4α expression in both crypt and surface epithelial cells (Figure 1B), as reported previously (Ahn et al., 2008; Darsigny et al., 2009; Chahar et al., 2014). In contrast, the isoform-specific antibodies reveal that P1-HNF4α is expressed mainly in the differentiated compartment, not in the proliferative compartment as defined by NKCC1 staining (Figure 1C). P2-HNF4α was observed primarily in the bottom half of the crypt (Figure 1B) and co-localized with the proliferation marker Ki67 in isolated colonic crypts (Figure 1D). While there was some expression of P2-HNF4α in the differentiated compartment (i.e., non Ki67 expressing cells), it was notably absent from the surface epithelium (Figure 1B). Figure 1 Download asset Open asset Differential localization of HNF4α isoforms in mouse colonic crypts. (A) Schematic of the mouse (and human) Hnf4a gene showing the two promoters (P1 and P2) (top) and the P1- and P2-driven HNF4α isoforms that they express (bottom). The differential N-terminal A/B domain (indicated in blue and orange) as well as epitopes to isoform-specific (αP1 and αP2) and common (αP1/P2) antibodies are indicated. DBD, DNA binding domain; LBD, ligand binding domain; F, F domain. (B–D) IF and immunohistochemistry of distal colon (B,C) or isolated colonic crypts (D) stained for the indicated proteins using the antibodies in (A) (B: 40X magnification; C,D: 25X magnification with digital zoom). NKCC1 (Slc12a2) (C) and Ki67 (D) mark the proliferative compartment of the crypt. Representative images from two independent experiments (n=2–4 mice per genotype) are shown. https://doi.org/10.7554/eLife.10903.003 Isoform-specific dysregulation of HNF4α in mouse models of colitis and colon cancer Previous studies showed that HNF4A expression is decreased in human inflammatory bowel disease (IBD) patients and intestine-specific deletion of the mouse Hnf4a gene increases susceptibility to dextran sodium sulfate (DSS)-induced colitis (Ahn et al., 2008) and can lead to chronic inflammation even in the absence of DSS (Darsigny et al., 2009). However, these studies do not address the role of the individual HNF4α isoforms. We treated young adult male mice (WT) with 2.5% DSS and found a statistically significant decrease in total HNF4α following 5 days of DSS treatment, as others have observed (Ahn et al., 2008; Chahar et al., 2014), and an increase in HNF4α during the recovery phase, especially P1-HNF4α (Figure 2A,B). Contrary to the restricted expression of P1-HNF4α in the differentiated compartment in untreated mice, P1-HNF4α was also expressed near the bottom of the crypt after DSS treatment (Figure 2C), consistent with substantial loss of proliferating cells following DSS treatment (Tessner et al., 1998). Figure 2 Download asset Open asset Dysregulation of P1- and P2-HNF4α in mouse models of colitis and colon cancer. (A) IB of WCE from the distal colon of WT mice treated with 2.5% DSS for 5 days followed by 0 or 3 days recovery, and an analogous region of untreated (Control) mice. Each lane is from a different mouse. The position of the molecular weight marker (52 kD) is shown. (B) Quantification of the HNF4α signal in (A) normalized to total protein, as determined by Coomassie staining of the same blot. For the purposes of quantification the outlier in lane 8 was omitted. *P<0.05, **P<0.005. (C) Representative IF of distal colon from untreated and DSS-treated WT mice (n=3–4 per condition) stained with P1-HNF4α antibody (40X magnification). Arrow indicates P1-HNF4α expressing cells near the bottom of the crypt in the DSS-treated mice. (D) IB as in (A) but from the tumor area of WT mice treated with 10 mg/kg AOM and three cycles of a 7-day DSS treatment and harvested at ~95 days. Three gels were run in parallel with the same extracts; one representative β-actin stain is shown. (E) IB analysis as in (D) but from mice injected six times with 10 mg/kg AOM and harvested at ~150 days. Shown is one representative of the three Coomassie stains performed for loading verification. https://doi.org/10.7554/eLife.10903.004 In a mouse model of colitis-associated colon cancer (CAC) in which a single injection of azoxymethane (AOM) is followed by multiple treatments of DSS in the drinking water, we found that P1-HNF4α is greatly reduced in tumors compared to untreated controls but that total HNF4α protein was only marginally reduced (Figure 2D), suggesting that P2-HNF4α was not affected. The P1-HNF4α decrease correlated with an increase in active Src (pSrc), consistent with our earlier finding that Src specifically phosphorylates and causes the degradation of human P1- but not P2-HNF4α (Chellappa et al., 2012). We also observed a specific loss of P1-HNF4α protein in a mouse model of sporadic, non-colitis colon cancer (Figure 2E), as we observed previously in humans (Chellappa et al., 2012). Differential susceptibility of HNF4α isoform-specific mice to colitis-associated colon cancer To decipher the function of the HNF4α isoforms in the colon, we utilized HNF4α isoform-specific mice generated by an exon swap strategy (Figure 3A top left) (Briançon and Weiss, 2006). These mice express exclusively either P1-HNF4α (α1HMZ) or P2-HNF4α (α7HMZ) wherever HNF4α is endogenously expressed. Immunoblot analysis confirmed that the HNF4α protein level in the distal colon of the exon swap mice is equivalent to that of WT littermates, and that P2-HNF4α is the major isoform in the distal colon (Figure 3A top right and Figure 3—figure supplement 1E). In α1HMZ mice, P1-HNF4α was detected in all epithelial cells in both the bottom of the crypt and the surface epithelium; a similar ubiquitous expression was observed for P2-HNF4α in α7HMZ mice (Figure 3A bottom). Figure 3 with 1 supplement see all Download asset Open asset Differential susceptibility of HNF4α isoform-specific mice to colitis-associated colon cancer. (A) Top left, Schematic of Hnf4a exon-swap (i.e., isoform-specific) mice. Top right, IB as in Figure 2A of WCE from the distal colon of the exon-swap mice and their WT controls, probed with the common αP1/P2 antibody. See Figure 3—figure supplement 1E for verification of protein loading. Bottom, representative IF of distal colons from untreated α1HMZ and α7HMZ mice stained with either P1- or P2-driven HNF4α specific antibodies (40X magnification). N=3–4 mice per genotype examined. Scale for P1-HNF4α α1HMZ is 0.22 x 0.22 microns; all others are 0.36 x 0.36 microns. (B) Tumor growth in WTα1 (n = 5) and α1HMZ (n = 6) mice treated with 10 mg/kg AOM and two cycles of DSS (5 days per cycle) and harvested at ~53 days. Right, number of tumors per mouse colon. Left, tumor load (sum of the width or length of all macroscopic lesions in a given mouse). Each symbol represents results from one mouse. (C) Left, average length of crypt in WTα1 and α1HMZ mice, untreated (Control) or treated as in (B) N = 2–3 mice per condition; 26–56 crypts per mouse were measured. *P<0.0005 between treated and control within a genotype and across genotypes in the treated condition. Right, Representative H&E stain (10X magnification) of mice treated as in (B) Scale bar is 100 microns. (D) Tumor number in WTα1 (n = 15) and α1HMZ (n = 17) male mice treated as in (B) but with three cycles of DSS (two cycles of 5 days and one cycle of 4-days) and harvested at ~85 days. Top, total number of tumors per mouse. Bottom, number of tumors per mouse based on the tumor width. n.s., non-significant. (E) As in (B) but for WTα7 (n = 21) and α7HMZ (n = 23) mice treated with 10 mg/kg AOM and 2–3 cycles of DSS (4–5 days per cycle) and harvested at ~53–64 days. P-values between α7HMZ and WTα7 mice are indicated. Tumor data were pooled from three independent experiments. (F) Tumor number and load in WTα7 (n = 20) and α7HMZ (n = 14) mice as in (E) but harvested at ~85 days. The following figure supplement is available for Figure 3: https://doi.org/10.7554/eLife.10903.005 After 53 days of AOM+DSS treatment, the α1HMZ mice had significantly fewer and smaller tumors compared to WT controls (Figure 3B). In addition, despite similar crypt length in untreated WT and α1HMZ mice, the α1HMZ mice did not exhibit the characteristic increase in crypt length associated with mutagen exposure observed in WT mice (Richards, 1977) (Figure 3C). In fact, the crypt length decreased compared to both treated WT and untreated α1HMZ. We also observed fewer infiltrating immune cells (Figure 3C right) as well as decreased spleen-to-body weight ratio in the treated α1HMZ mice (Figure 3—figure supplement 1A). After 85 days of treatment, the difference in tumor number was less pronounced: a significant decrease in tumor number was observed in α1HMZ mice only in the smallest tumors (0–2 mm) (Figure 3D). In contrast to α1HMZ mice, the α7HMZ mice exhibited a greater tumor load and tumor number than their WT controls after 53–64 days of treatment (Figure 3E). However, at the later time point (85 days), the effect was lost mainly due to increased tumor burden in the WT mice (Figure 3F and Figure 3—figure supplement 1B). Interestingly, there was no difference in the percent of Ki67-staining cells between WT and α7HMZ mice (53–64 day treatment) (Figure 3—figure supplement 1C,D). Differential susceptibility of HNF4α isoform-specific mice to colitis More striking than tumor induction by AOM+DSS in the α1HMZ and α7HMZ mice was their response to an acute DSS treatment to induce colitis --2.5% DSS in drinking water for 5 days. There was a ~73% mortality rate for α7HMZ mice that occurred starting after three days of recovery when the mice were switched to normal tap water (Figure 4A). During the recovery phase, α7HMZ mice exhibited a significant decrease in body weight and colon length (Figure 4B and Figure 4—figure supplement 1A), and a worse histological score (due to more severe crypt damage, inflammation and ulceration) compared to their WT littermates (Figure 4C and Figure 4—figure supplement 1B). There was also an increased spleen-to-body weight ratio (Figure 4—figure supplement 1C) when the mice were maintained and treated in an open access vivarium. IB analysis revealed that, in contrast to the WT mice that lost expression of both HNF4α isoforms after five days of DSS and then had an increase in P1-HNF4α expression at 3-day recovery (Figure 2A), in the α7HMZ mice P2-HNF4α protein amount is notably increased upon DSS treatment and then decreased after a 3-day recovery, as observed by both IB and IF (Figure 4D). At 12 days of recovery, we observed a massive infiltration of immune cells and a continued striking loss in crypt structure in α7HMZ mice compared to WT mice (Figure 4—figure supplement 1D). Figure 4 with 2 supplements see all Download asset Open asset Differential susceptibility of HNF4α isoform-specific mice to DSS-induced colitis. (A) Percent mortality of WTα7 (n = 28) and α7HMZ (n = 16) mice treated with 2.5% DSS for 5 days. α7HMZ mice typically died during day 3 to 12 of recovery following DSS treatment. Data pooled from two independent experiments. Not shown is a third experiment with older mice (21–23 weeks) with similar results (WT: 1 of 5 mice died; α7HMZ: 3 of 6 mice died). (B) Change in bodyweight (represented as% initial body weight) (left) and colon length (right) of WT (n = 4) and α7HMZ (n = 4) mice treated as indicated. Significant comparisons are indicated with a P-value. (C) Left, representative H&E stain of WT and α7HMZ mice treated with 2.5% DSS for 5 days followed by 0 or 3 days of recovery. Right, histological scores of colitis in WTα7 (n = 4) and α7HMZ (n = 4) mice. (D) Left, IB for HNF4α (P1/P2 antibody) of WCE from the distal colon of α7HMZ mice treated as indicated. Right, representative IF of distal colon from α7HMZ mice treated with 2.5% DSS for 5 days -/+ recovery as indicated and stained with P1/P2-HNF4α antibody (green) and TO-PRO3 (red) for nuclei (40X magnification). Extracts from four mice per genotype (out of n = 5–7) were randomly chosen for IB analysis on a single gel/blot; sections from 3 mice per genotype were examined. (E) Colon length of WT (n = 8) and α1HMZ (n = 10) male mice treated with 2.5% DSS for 5 days followed by 3 days of recovery. Results from two independent experiments were pooled. (F) Representative H&E stain (left) and histological scores (right) of colitis in WTα1 (n = 8) and α1HMZ (n = 10) mice treated as in (E). The following figure supplements are available for Figure 4: https://doi.org/10.7554/eLife.10903.007 In contrast to the extreme sensitivity of the α7HMZ mice to DSS-induced colitis, α1HMZ mice were less susceptible than their WT controls as indicated by increased colon length (Figure 4E) and well-preserved crypt structure and decreased histological score (Figure 4F). There was no difference in spleen-to-body weight ratio between the α1HMZ mice and WT controls (data not shown). Clinical and histological changes occurring a few weeks after DSS treatment are referred to as chronic or advanced changes (Perše and Cerar, 2012). To examine chronic effects, we allowed the mice to recover for 18 days after a somewhat milder DSS treatment (4 days) to reduce the mortality of α7HMZ mice. Despite the shorter DSS treatment, after 18 days, the α7HMZ mice still exhibited elevated spleen-to-body weight ratio, increased crypt length, more visibly inflamed colons and immune cell infiltration and overall higher histological scores compared to α1HMZ mice (Figure 4—figure supplement 2A–D). Transcriptomic and proteomic profile of colons from HNF4α isoform-specific mice Expression profiling of the distal colon revealed a significant change in a substantial number of genes in the untreated isoform-specific mice compared to their WT controls (Figure 5—figure supplement 1A). There was an overall greater effect in α1HMZ than α7HMZ mice in terms of the number of dysregulated genes with a large fold change, which could be due to the fact that P1-HNF4α typically has a more potent transactivation function than P2-HNF4α (Eeckhoute et al., 2003). On the other hand the number of genes altered at lower fold change was higher in α7HMZ compared to α1HMZ mice, consistent with more P2-HNF4α protein in the distal colon of WT mice than P1-HNF4α (Figure 3A) . Gene Ontology (GO) analysis of the differentially regulated genes showed that in α1HMZ mice there is a marked upregulation of genes involved in wound healing and immune response, as well as a variety of metabolic processes typically associated with differentiation (Figure 5A). In contrast, in α7HMZ mice there is a significant upregulation of genes involved in cell cycle and DNA repair and a decrease in genes involved in cell adhesion, motility and ion transport (Figure 5B). (See Figure 5—source data 1A-1G for the fold change in the top 100 dysregulated genes and the genes in the aforementioned GO categories, respectively). Figure 5 with 1 supplement see all Download asset Open asset Altered gene expression, interacting proteins, migration and ion transport in HNF4α isoform-specific mice. (A,B) Comparative Gene Ontology (GO) of genes differentially regulated (≥two-fold) in the distal colon of untreated α1HMZ (A) and α7HMZ (B) mice. (C) Top, Venn diagram of total number of HNF4α-interacting proteins from RIME analysis found in α7HMZ only, α1HMZ only or both α7HMZ and α1HMZ colons, as described in Material and methods. Indicated are nuclear proteins that have been implicated in regulating gene expression and associated with human or mouse colon cancer, IBD, Crohn’s disease and/or ulcerative colitis, as well as other pro-proliferative proteins found only in α7HMZ colons. Shown also are transcription factors that interact with HNF4α in both genotypes. Bold, proteins mentioned in text. Bottom, Total number of proteins in the indicated categories that show a significant interaction with HNF4α in the exon swap mice. TF, transcription factor; RNA binding proteins; kinase and phosphatase categories include only protein kinases and phosphatases, as well as relevant scaffolding proteins b. (D) Untreated HNF4α isoform-specific mice and their WT littermates (n = 3–4 per genotype) were injected with BrdU (75 mg/kg) and sacrificed at 2 hr or 48–50 hr. The distance migrated by the BrdU+ cells from the bottom of the crypt between 2 hr and 48–50 hr is plotted as% crypt length; 5–38 crypts per mouse were scored. (E) Intestinal chloride secretion in response to 10 µM forskolin and 100 µM carbachol represented as change in short-circuit current (ΔIsc). Left, WTα1 (n = 6) and α1HMZ (n = 5–8) mice. *P<0.02 between α1HMZ and WTα1. Right, WTα7 (n = 4) and α7HMZ (n = 3) mice. *P<0.05 versus WTα7. Results from one experiment per genotype are shown: a second independent experiment for α7HMZ yielded similar results (not shown). (F) Left, RELMβ mRNA expression in the distal colon of untreated α1HMZ, α7HMZ and their WT controls from microarrays in (A,B), represented as an average of the three Retnlb probes. P<0.008 versus WTα7. Right, RELMβ protein level quantified by ELISA in the mid colon homogenate of untreated WTα7 (n = 5) and α7HMZ (n = 3) mice. Shown are means of technical triplicates. The following supplementary figure and source data are available for Figure 5: https://doi.org/10.7554/eLife.10903.010 Figure 5—source data 1 Transcriptomic analysis of HNF4α isoform-specific mice. (A) Top 100 genes DOWN in distal colon of α1HMZ male mice compared to WT controls. (B) Top 100 genes UP in distal colon of α1HMZ male mice compared to WT controls. (C) Top 100 genes DOWN in distal colon of α7HMZ male mice compared to WT controls. (D) Top 100 genes UP in distal colon of α7HMZ male mice compared to WT controls. (E) Up-regulated genes involved in wound healing and immune function enriched in α1HMZ mice. (F) Up-regulated genes involved in cell cycle and DNA repair in α7HMZ mice. (G) Down-regulated genes involved in cell adhesion and ion transport in α7HMZ mice. https://doi.org/10.7554/eLife.10903.011 Download elife-10903-fig5-data1-v3.xlsx Figure 5—source data 2 Proteomic analysis of HNF4α isoform-specific mice. (A) List of proteins that interact with HNF4α in α1HMZ and α7HMZ colons from RIME analysis meeting the criteria described in Figure 5C. (B) Select proteins that interact with HNF4α in α1HMZ and α7HMZ colons from RIME analysis used to prepare the graph in Figure 5C. (C) All peptides that interact with HNF4α in both α7HMZ and α1HMZ colons from RIME analysis in which there are at least 2 positives for each genotype. (D) All peptides that interact with HNF4α preferentially in α7HMZ colons in which there are 2 or more positives for α7HMZ compared to α1HMZ. (E) All peptides that interact with HNF4α preferentially in α1HMZ colons in which there are 2 or more positives for α1HMZ compared to α7HMZ. Figure 5C. https://doi.org/10.7554/eLife.10903.012 Download elife-10903-fig5-data2-v3.xlsx The DNA binding domains of P1- and P2-HNF4α are 100% identical and the isoforms have similar in vitro DNA binding specificity and chromatin immunoprecipitation (ChIP)-seq profiles in human colon cancer cells (Vuong et al., 2015). Therefore, to elucidate the mechanism responsible for differential gene expression in mouse colon, we performed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) on HNF4α in the colons of α1HMZ and α7HMZ mice (Figure 5C). The isoforms share 76 interacting proteins, including previously reported HNF4γ (Daigo et al., 2011), a well known co-regulator for nuclear receptors (NRIP1, RIP140) and DPF2, a BRG1-associated factor (BAF45). However, there were more proteins uniquely binding HNF4α in α7HMZ and α1HMZ colons -- 138 and 99, respectively (Figure 5C top and Figure 5C—source data 2A–E). Src tyrosine kinase, for example, bound uniquely in α1HMZ colons, consistent with our previous report that Src preferentially phosphorylates and interacts with HNF4α1 in cell-based and in vitro systems (Chellappa et al., 2012) and validating RIME for identification of differential interacting proteins in vivo. In contrast, CUL4A, a core component of a cullin-based E3 ubiquitin ligase complex and overexpressed in cancer (Kopanja et al., 2009), and PCM1, a centrosome binding protein translocated to the JAK2 locus in certain leukemias (Reiter et al., 2005), both bound uniquely in α7HMZ colons. Both CUL4A and PCM1 are required for efficient cell proliferation, genome stability and/or proper centrosome function (Erger and Casale, 1998; Farina et al., 2016), consistent with the upregulation of genes involved in cell cycle and DNA repair in α7HMZ colons (Figure 5B), and accelerated tumorigenesis in α7HMZ mice (Figure 3E). Cross-referencing the interacting proteins to those in the literature associated with colon cancer and inflammatory bowel disease (IBD) revealed several additional relevant proteins for each genotype, t

The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways.

Human lymphoblastoid cell lines (LCLs), generated through Epstein-Barr Virus (EBV) transformation of B-lymphocytes (B-cells), are a commonly used model system for identifying genetic influences on human diseases and on drug responses. We have previously used LCLs to examine the cellular effects of genetic variants that modulate the efficacy of statins, the most prescribed class of cholesterol-lowering drugs used for the prevention and treatment of cardiovascular disease. However, statin-induced gene expression differences observed in LCLs may be influenced by their transformation, and thus differ from those observed in native B-cells. To assess this possibility, we prepared LCLs and purified B-cells from the same donors, and compared mRNA profiles after 24 h incubation with simvastatin (2 µm) or sham buffer. Genes involved in cholesterol metabolism were similarly regulated between the two cell types under both the statin and sham-treated conditions, and the statin-induced changes were significantly correlated. Genes whose expression differed between the native and transformed cells were primarily implicated in cell cycle, apoptosis and alternative splicing. We found that ChIP-seq signals for MYC and EBNA2 (an EBV transcriptional co-activator) were significantly enriched in the promoters of genes up-regulated in the LCLs compared with the B-cells, and could be involved in the regulation of cell cycle and alternative splicing. Taken together, the results support the use of LCLs for the study of statin effects on cholesterol metabolism, but suggest that drug effects on cell cycle, apoptosis and alternative splicing may be affected by EBV transformation. This dataset is now uploaded to GEO at the accession number GSE51444.

BACKGROUND:Statins are widely prescribed for lowering LDL-cholesterol (LDLC) levels and risk of cardiovascular disease. There is, however, substantial inter-individual variation in the magnitude of statin-induced LDLC reduction. To date, analysis of individual DNA sequence variants has explained only a small proportion of this variability. The present study was aimed at assessing whether transcriptomic analyses could be used to identify additional genetic contributions to inter-individual differences in statin efficacy.RESULTS:Using expression array data from immortalized lymphoblastoid cell lines derived from 372 participants of the Cholesterol and Pharmacogenetics clinical trial, we identify 100 signature genes differentiating high versus low statin responders. A radial-basis support vector machine prediction model of these signature genes explains 12.3% of the variance in statin-mediated LDLC change. Addition of SNPs either associated with expression levels of the signature genes (eQTLs) or previously reported to be associated with statin response in genome-wide association studies results in a combined model that predicts 15.0% of the variance. Notably, a model of the signature gene associated eQTLs alone explains up to 17.2% of the variance in the tails of a separate subset of the Cholesterol and Pharmacogenetics population. Furthermore, using a support vector machine classification model, we classify the most extreme 15% of high and low responders with high accuracy.CONCLUSIONS:These results demonstrate that transcriptomic information can explain a substantial proportion of the variance in LDLC response to statin treatment, and suggest that this may provide a framework for identifying novel pathways that influence cholesterol metabolism.

Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs—HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2—reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ∼100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe .

Genes Cluster ID name T6/T3 T8/T3 T11/T3 T24/T3 gene name function CIBE_0313 6up++ 5.3 6.2 7.2 5.8 conserved protein of unknown function CIBE_0312 6up++ 5.0 6.3 7.4 5.6 conserved protein of unknown function CIBE_0311 6up++ 5.0 6.4 7.0 5.3 conserved protein of unknown function CIBE_0314 6up++ 4.1 5.9 7.0 5.8 conserved protein of unknown function CIBE_0315 6up++ 5.1 5.9 6.1 5.0 Radical SAM-family protein CIBE_0310 6up++ 4.7 6.3 5.9 4.6 putative ABC transporter ATP-binding protein CIBE_3477 6up++ 5.5 5.6 5.4 4.4 Accessory gene regulator B CIBE_3478 6up++ 5.3 5.8 5.0 4.3 conserved membrane protein of unknown function CIBE_0316 6up++ 4.5 5.5 5.6 4.2 conserved protein of unknown function CIBE_4430 6up++ 4.4 6.4 5.4 3.6 putative permease CIBE_4429 6up++ 3.7 5.7 4.8 3.5 putative permease CIBE_0280 6up++ 4.5 4.9 4.3 3.9 conserved exported protein of unknown function CIBE_3828 6up++ 4.4 5.6 4.4 3.2 Glycosyl transferase CIBE_4431 6up++ 2.7 5.4 5.5 3.6 conserved exported protein of unknown function CIBE_4419 6up++ 3.7 5.5 4.4 3.1 conserved exported protein of unknown function CIBE_4428 6up++ 3.1 5.4 4.7 3.2 glr Transporter CIBE_4418 6up++ 3.4 5.4 4.4 2.4 conserved exported protein of unknown function CIBE_3476 6up+ 4.4 4.6 4.3 3.6 conserved protein of unknown function CIBE_3751 6up+ 3.8 5.2 3.8 3.1 conserved protein of unknown function CIBE_3750 6up+ 3.9 4.6 3.7 3.4 conserved protein of unknown function CIBE_4789 6up+ 3.7 4.3 3.9 3.5 cfg putative AgrB-like protein CIBE_0824 6up+ 4.8 3.9 3.8 2.7 Type 11 methyltransferase CIBE_3749 6up+ 4.1 4.4 3.5 2.8 Peptide synthetase CIBE_5303 6up+ 4.6 4.0 3.1 2.7 conserved protein of unknown function CIBE_2622 6up+ 2.8 3.7 3.9 4.0 adh NADPH-dependent butanol dehydrogenase CIBE_4040 6up+ 3.0 3.3 4.1 3.7 Aliphatic sulfonates family ABC transporter, periplsmic ligand-binding protein CIBE_3079 6up+ 4.3 3.5 3.3 2.8 panD aspartate 1-decarboxylase CIBE_4039 6up+ 2.9 3.2 3.9 4.1 tauB taurine transporter subunit ; ATP-binding component of ABC superfamily CIBE_3082 6up+ 4.2 3.4 3.0 2.9 conserved protein of unknown function CIBE_4610 6up+ 2.1 4.1 4.1 3.3 Peptidase C1A, papain CIBE_4417 6up+ 3.1 4.7 3.5 2.2 conserved membrane protein of unknown function CIBE_3475 6up+ 3.5 3.7 3.4 2.8 Histidine kinase CIBE_4036 6up+ 2.7 3.0 3.9 3.7 Thioredoxin reductase CIBE_3355 6up+ 3.8 3.0 3.1 3.3 RNA polymerase sigma factor SigI CIBE_3081 6up+ 4.1 3.4 2.9 2.8 panB ketopantoate hydroxymethyltransferase CIBE_4038 6up+ 2.8 2.8 3.8 3.7 Type I phosphodiesterase/nucleotide pyrophosphatase CIBE_2623 6up+ 2.6 3.3 3.8 3.3 conserved protein of unknown function CIBE_5304 6up+ 4.3 3.6 2.7 2.4 conserved protein of unknown function CIBE_3537 6up+ 2.9 4.5 2.5 3.1 putative transcriptional regulator CIBE_5707 6up+ 3.0 3.4 3.6 2.9 conserved protein of unknown function CIBE_0782 6up+ 2.2 2.8 3.7 4.1 Nitrogenase CIBE_4041 6up+ 2.3 3.1 3.8 3.6 tauC taurine transporter subunit ; membrane component of ABC superfamily CIBE_2624 6up+ 2.2 3.3 3.8 3.5 sudA Sulfide dehydrogenase subunit alpha CIBE_3080 6up+ 4.0 3.3 3.1 2.3 panC pantothenate synthetase CIBE_3353 6up+ 3.5 2.7 2.7 3.4 Beta-glucanase (fragment) CIBE_4861 6up+ 1.9 3.8 3.2 3.4 conserved protein of unknown function CIBE_3700 6up+ 1.2 3.3 3.9 3.9 YIEGIA protein CIBE_3753 6up+ 3.1 3.6 2.4 3.1 protein of unknown function CIBE_3354 6up+ 3.8 2.9 2.6 2.8 bglA Beta-glucanase CIBE_3748 6up+ 3.7 3.7 2.7 2.0 putative cyclic peptide transporter CIBE_4416 6up+ 3.2 4.3 3.2 1.2 conserved membrane protein of unknown function CIBE_1176 6up+ 4.3 3.3 2.6 1.7 rplY 50S ribosomal protein L25 CIBE_4788 6up+ 3.2 3.5 2.6 2.4 CHAP domain containing protein CIBE_3357 6up+ 4.2 2.8 2.3 2.5 Carbohydrate-binding family V/XII CIBE_2891 6up+ 3.7 3.1 2.3 2.6 Electron transport complex, RnfABCDGE type, D subunit CIBE_6013 6up+ 4.4 3.5 1.9 1.7 conserved protein of unknown function CIBE_0281 6up+ 3.7 3.9 3.3 0.7 conserved membrane protein of unknown function CIBE_4862 6up+ 1.8 3.3 3.1 3.3 2-hydroxyglutaryl-CoA dehydratase CIBE_5605 6up+ 2.2 2.8 3.5 3.0 conserved protein of unknown function CIBE_2892 6up+ 3.8 3.0 2.2 2.3 Electron transport complex, RnfABCDGE type, G subunit CIBE_5208 6up+ 3.0 2.9 3.1 2.4 Nucleotidyl transferase CIBE_0725 6up+ 2.5 2.9 3.1 2.5 cstA carbon starvation-induced membrane protein CIBE_3837 6up+ 3.0 3.7 2.1 2.2 yqaJ putative nuclease; skin element CIBE_4414 6up+ 2.7 3.7 2.8 1.7 Cell wall-associated hydrolase-like protein CIBE_4037 6up+ 2.2 2.3 3.5 2.8 Thioredoxin domain CIBE_5606 6up+ 2.2 2.7 3.2 2.8 Membrane spanning protein CIBE_3761 6up+ 3.7 2.9 2.4 1.4 Multidrug transporter MatE CIBE_3441 6up+ 1.6 3.4 3.1 2.4 ABC transporter substrate-binding protein CIBE_2221 6up+ 1.1 3.7 3.5 2.0 ytfJ Uncharacterized spore protein YtfJ CIBE_2951 6up+ 1.4 3.5 2.8 2.3 conserved protein of unknown function CIBE_2222 6up+ 1.4 3.5 3.0 1.6 conserved membrane protein of unknown function CIBE_3534 6up+ 1.4 3.3 2.3 2.0 putative DnaD-like protein, phage replisome organizer functional annotation genes

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/ tfe.

The epigenetic activator Mixed lineage leukemia 1 (MLL1) is paramount for embryonic development and hematopoiesis. Here, we demonstrate that the long, noncoding RNA (lncRNA) Mistral (Mira) activates transcription of the homeotic genes Hoxa6 and Hoxa7 in mouse embryonic stem cells (mESC) by recruiting MLL1 to chromatin. The Mira gene is located in the spacer DNA region (SDR) separating Hoxa6 and Hoxa7, transcriptionally silent in mESCs, and activated by retinoic acid. Mira-mediated recruitment of MLL1 to the Mira gene triggers dynamic changes in chromosome conformation, culminating in activation of Hoxa6 and Hoxa7 transcription. Hoxa6 and Hoxa7 activate the expression of genes involved in germ layer specification during mESC differentiation in a cooperative and redundant fashion. Our results connect the lncRNA Mira with the recruitment of MLL1 to target genes and implicate lncRNAs in epigenetic activation of gene expression during vertebrate cell-fate determination.

Background: Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression. Results: Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4a, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4a binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4a binds Alu elements in the promoters of target genes (ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR) and luciferase assays to show that at least some of those Alu elements can modulate HNF4a-mediated transactivation in vivo (APOM, PRODH2, TTR, APOA4). HNF4a-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4a binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4a sites. Conclusions: Our findings suggest that HNF4a, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats. Background As much as 50% of the ~3 billion base pairs in the human genome may be derived from repetitive DNA sequence [1]. While repetitive DNA is often referred to as “junk” DNA, even when that term was originally coined it was hypothesized that junk DNA may play an active role in genome function [2]. The notion that repetitive DNA may play a regulatory role and be involved in the evolution of gene regulation was also postulated early on, although it was not until recently that there was evidence to support those ideas [3-5]. A major category of repetitive DNA is short interspersed nuclear elements (SINEs), which are believed to have originated from the 7SL RNA gene that is part of the ribosome complex [6]. In the human genome, the largest class of SINEs are Alu repeats, which at ~1.2 million copies account for ~10% of the human genome [1]. Alu elements were first characterized as ~300 nucleotide repetitive sequences that contain an AluI restriction site (5’-AGCT-3’) from the bacterium Arthrobacter luteus [7,8]. Alu elements, which are still mobile in the human genome by virtue of the action of a LINE1 reverse transcriptase [9], are a relatively recent occurrence evolutionarily. They are found exclusively in primates, including humans, and hence are postulated to have entered the mammalian genome ~60-65 million years ago [10]. Alu elements have been implicated in several human diseases including leukemia, hemophilia and breast cancer, suggesting that their impact on human health may be significant [11]. There are several well characterized examples of Alu insertions affecting splicing patterns and hence protein function [12]. A variety of transcription factor (TF) binding sites (TFBSs) have also been * Correspondence: frances.sladek@ucr.edu Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA, 92521, USA Full list of author information is available at the end of the article Bolotin et al. BMC Genomics 2011, 12:560 http://www.biomedcentral.com/1471-2164/12/560 © 2011 Bolotin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. characterized in Alu elements, including sites for YY1 [13], Sp1 [14], tumor suppressor p53 [15], homeodomain and TATA binding proteins [16]. Nuclear receptors (NR), which belong to a superfamily of liganddependent TFs, have also been found to have binding sites in Alu elements: retinoid acid receptor (RAR, NR1B) [17], estrogen receptor (ER, NR3A) [18,19], progesterone receptor (PR, NR3C3) [20] and vitamin D receptor (VDR, NR1I1) [21]. Alu insertions have also been shown to alter the expression of at least six human genes: CD8a (CD8A), keratin 18 (KRT18), parathyroid hormone (PTH), Wilm’s tumor 1 (WT1), receptor for Fc fragment of IgE, high affinity I, gamma polypeptide (FCER1G) and breast cancer 1, early onset (BRCA1) [22]. Therefore, Alu sequences may regulate the level of transcripts and hence proteins in the cell, as well as the function of those proteins. Hepatocyte nuclear factor 4 alpha, (HNF4a, NR2A1) is a member of the NR superfamily that is highly expressed in the liver, as well as the kidney, intestine (large and small), pancreas and stomach [23]. HNF4a is best known for its role in the adult liver and pancreas, as well as in early development [24,25]; it also has an emerging role in the gut [26-28]. The HNF4Α gene is mutated in an inherited form of type 2 diabetes, maturity onset diabetes of the young 1 (MODY1) [29], and was recently identified as a susceptibility locus in inflammatory bowel disease (IBD) [30]. Mutations in HNF4a binding sites have also been directly linked to human diseases, including hemophilia and MODY3 [31,32]. Many NRs are common drug targets [33]; the recent identification of the endogenous ligand of HNF4a that binds in a reversible fashion also makes HNF4a a potential drug target [34,35]. In addition to its medical relevance, HNF4a also appears to play a unique role in the evolution of NRs. It is highly conserved across species, with 100% amino acid conservation in the DNA binding domain of all mammalian HNF4a. While HNF4a is most similar to the retinoid × receptor alpha (RXRa, NR2B1), unlike many other NRs, it does not heterodimerize with RXR. Rather, it binds DNA in the form of direct repeats separated by one nucleotide (DR1, AGGTCAxAGGTCA) exclusively as a homodimer [36]. HNF4a has been found in every animal organism examined thus far, including sponge and coral [37], and has been postulated to be the ancestor of the entire NR family [38]. Many hundreds of HNF4a target genes have been identified by both classical promoter analysis as well as more modern genome-wide studies [32,39-41]. During one such genomic study, we observed a very uneven frequency profile of individual HNF4a binding sequences [42]. Specifically, we noted that a certain DNA sequence designated H4.141 (5’-AGGCTGaAGTGCA-3’) was > 100-fold overrepresented compared to other HNF4a binding sites in the human, but not the mouse, genome (see additional file 1: Figure S1). In the current study, we investigate the notion that these and other HNF4a binding sequences are in Alu repeats. We use the powerful high throughput technology of protein binding micorarrays (PBMs) to show that HNF4a does indeed bind numerous sequences in Alu repeats in vitro. We perform ChIP and luciferase assays to show that HNF4a binds at least some Alu sequences in vivo and that those binding events are associated with transcriptional activation. Finally, we investigate accessibility of these sites by correlation with DNase hypsersensitivity data and evolutionary conservation by comparative genomic analysis. Results HNF4a binds Alu repeats in vitro Since genome-wide location analysis (i.e., ChIP-chip/seq) often filters out or cannot distinguish the exact location of TF binding events in highly repetitive DNA, we took a combined in vitro/in silico approach to determine whether HNF4a binds Alu elements. We generated a custom protein binding microarray (PBM3) that contained 200 unique Alu-associated sequences (Figure 1). Since RAR was previously shown to bind DR2-like sequences (AGGTCAxxAGGTCA) in Alu repeats [17] and since we have previously shown that HNF4a, while preferring DR1s, can also bind DR2s [43], we also put on the PBM ~1470 permutations of DR1 and DR2 sequences as well as ~150 random controls and ~2000 additional sequences in the human genome predicted by a support vector machine (SVM) algorithm to bind HNF4a [42]. Each sequence was replicated four times for a total of more than 15,000 spots of DNA. We found that human HNF4a2 bound 66 out of 200 Alu-derived 13-mers in a significant fashion (> 2 SD better than random controls, p-value < 0.045 for the lowest binder) (Figure 2A). It also bound 994 out of 3796 non Alu-derived sequences, although eight of those sequences were subsequently found also to be associated with Alu repeats at a frequency of > 90%. An exact match search of the entire human genome (hg18) with the 1060 sequences that bound HNF4a in the PBM (66 + 994) showed that there are a total of 1,320,513 occurrences of those HNF4a binding sites in the genome and that the vast majority (94.9%, 1,252,918) are in repetitive elements, of which most (95.7%, 1,198,534) are in Alu repeats (Figure 2A). This number is much greater than that previously found for RAR binding sites in Alu elements but that is most likely due to the fact that strict DR1 and DR2 consensus sequences were used for the genomic search [17]. Bolotin et al. BMC Genomics 2011, 12:560 http://www.biomedcentral.com/1471-2164/12/560 Page 2 of 15

Background Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression. Results Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes ( ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR ) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation in vivo ( APOM, PRODH2, TTR, APOA4 ). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites. Conclusions Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.