Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer

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. DOI: http://dx.doi.org/10.7554/eLife.10903.001


Introduction
Hepatocyte nuclear factor 4alpha (HNF4a) (NR2A1) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver, kidney, pancreas, stomach and intestine . HNF4a 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). HNF4a is also known for its role in the pancreas where it regulates insulin secretion from beta cells (Gupta et al., 2007;Miura et al., 2006). Mutations in the HNF4A gene and promoter regions are associated with Maturity Onset Diabetes of the Young 1 (MODY1) (Ellard and Colclough, 2006).
In contrast, the role of HNF4a 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 HNF4a 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 HNF4a 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 HNF4a   (Figure 1A). The major isoforms of the P1 promoter are HNF4a1/a2 while the P2 promoter gives rise to HNF4a7/a8: 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-HNF4a (Tanaka et al., 2006;Nakhei et al., 1998). While a loss of P1-but not P2-HNF4a has been noted in colon cancer Tanaka et al., 2006), the specific roles of the HNF4a isoforms remain obscure. For example, P1-driven HNF4a 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-HNF4a 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 HNF4a isoforms may indeed play distinct roles in the colon.
Here, we investigate the role of P1-and P2-HNF4a isoforms in the mouse colon using genetically engineered mice that express either the P1-or the P2-HNF4a isoforms (Brianç on and Weiss, 2006). We show that in wildtype (WT) mice P1-and P2-HNF4a are expressed in different compartments in 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 HNF4a. In various diseases of the digestive system, such as gastric cancer or inflammatory bowel disease, the production of HNF4a is not properly regulated.
Gene expression can be activated by transcription factors binding to regions of DNA called promoters. The gene that encodes HNF4a has two promoters called P1 and P2, and each produce several different versions of the HNF4a 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 HNF4a, the structure of the crypts is disrupted. By studying crypts taken from the colon of mice, Chellappa et al. have now found that P1-HNF4a proteins are mainly produced at the top of the crypts, whereas P2-HNF4a proteins are found mainly at the bottom.
Chellappa et al. then used two sets of genetically engineered mice: one that can only produce P1-HNFa proteins, and one that only has P2-HNFa 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-HNF4a proteins were less susceptible to colitis and got fewer and smaller tumors than normal mice. By contrast, mice that could only produce P2-HNF4a 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 RELMb was produced in P2-only mice. Chellappa et al. then proceeded to show that RELMb is essential for the susceptibility of P2-mice to coliltis.
Overall, the experiments show that P1-HNF4a and P2-HNF4a 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. 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 RELMb, a member of the RELM/FIZZ family of cytokines, plays a critical role in the response of HNF4a to colitis and appears to be both directly and indirectly regulated by HNF4a.

Results
Compartmentalization of P1-and P2-HNF4a 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 HNF4a isoforms Tanaka et al., 2006) ( Figure 1A) to examine the distribution of P1-and P2-HNF4a along the crypt-surface axis. The P1/P2 antibody, which recognizes both P1-and P2-HNF4a, shows HNF4a 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-HNF4a is expressed mainly in the differentiated compartment, not in the proliferative compartment as defined by NKCC1 staining ( Figure 1C). P2-HNF4a 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-HNF4a in the differentiated compartment (i.e., non Ki67 expressing cells), it was notably absent from the surface epithelium ( Figure 1B).

Isoform-specific dysregulation of HNF4a 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 HNF4a isoforms. We treated young adult male mice (WT) with 2.5% DSS and found a statistically significant decrease in total HNF4a following 5 days of DSS treatment, as others have observed (Ahn et al., 2008;Chahar et al., 2014), and an increase in HNF4a during the recovery phase, especially P1-HNF4a (Figure 2A,B). Contrary to the restricted expression of P1-HNF4a in the differentiated compartment in untreated mice, P1-HNF4a 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).
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-HNF4a is greatly reduced in tumors compared to untreated controls but that total HNF4a protein was only marginally reduced ( Figure 2D), suggesting that P2-HNF4a was not affected. The P1-HNF4a 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-HNF4a . We also observed a specific loss of P1-HNF4a protein in a mouse model of sporadic, non-colitis colon cancer ( Figure 2E), as we observed previously in humans .
Differential susceptibility of HNF4a isoform-specific mice to colitisassociated colon cancer To decipher the function of the HNF4a isoforms in the colon, we utilized HNF4a isoform-specific mice generated by an exon swap strategy ( Figure 3A top left) (Brianç on and Weiss, 2006). These mice express exclusively either P1-HNF4a (a1HMZ) or P2-HNF4a (a7HMZ) wherever HNF4a is endogenously expressed. Immunoblot analysis confirmed that the HNF4a protein level in the distal colon of the exon swap mice is equivalent to that of WT littermates, and that P2-HNF4a is the major isoform in the distal colon ( Figure 3A  After 53 days of AOM+DSS treatment, the a1HMZ mice had significantly fewer and smaller tumors compared to WT controls ( Figure 3B). In addition, despite similar crypt length in untreated WT and a1HMZ mice, the a1HMZ 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 a1HMZ. We also observed fewer infiltrating immune cells ( Figure 3C right) as well as decreased spleen-to-body weight ratio in the treated a1HMZ 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 a1HMZ mice only in the smallest tumors (0-2 mm) ( Figure 3D).
In contrast to a1HMZ mice, the a7HMZ 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  Differential susceptibility of HNF4a isoform-specific mice to colitis More striking than tumor induction by AOM+DSS in the a1HMZ and a7HMZ 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 a7HMZ mice that occurred starting after three days of recovery when the mice were switched to normal tap water ( Figure 4A). During the recovery phase, a7HMZ mice  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 HNF4a isoforms after five days of DSS and then had an increase in P1-HNF4a expression at 3-day recovery (Figure 2A), in the a7HMZ mice P2-HNF4a 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 a7HMZ mice compared to WT mice (Figure 4-figure supplement 1D).
In contrast to the extreme sensitivity of the a7HMZ mice to DSS-induced colitis, a1HMZ 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 a1HMZ 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 (Persˇe 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 a7HMZ mice. Despite the shorter DSS treatment, after 18 days, the a7HMZ 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 a1HMZ mice ( Transcriptomic and proteomic profile of colons from HNF4a isoformspecific 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 a1HMZ than a7HMZ mice in terms of the number of dysregulated genes with a large fold change, which could be due to the fact that P1-HNF4a typically has a more potent transactivation function than P2-HNF4a (Eeckhoute et al., 2003). On the other hand the number of genes altered at lower fold change was higher in a7HMZ compared to a1HMZ mice, consistent with more P2-HNF4a protein in the distal colon of WT mice than P1-HNF4a ( Figure 3A) . Gene Ontology (GO) analysis of the differentially regulated genes showed that in a1HMZ 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 a7HMZ 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).
The DNA binding domains of P1-and P2-HNF4a 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 HNF4a in the colons of a1HMZ and a7HMZ mice ( Figure 5C).

Figure 3 continued
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 WTa7 (n = 21) and a7HMZ (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 a7HMZ and WTa7 mice are indicated. Tumor data were pooled from three independent experiments. (F) Tumor number and load in WTa7 (n = 20) and a7HMZ (n = 14) mice as in (E) but harvested at~85 days. The following figure supplement is available for Figure   . Differential susceptibility of HNF4a isoform-specific mice to DSS-induced colitis. (A) Percent mortality of WTa7 (n = 28) and a7HMZ (n = 16) mice treated with 2.5% DSS for 5 days. a7HMZ 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; a7HMZ: 3 of 6 mice died). (B) Change in bodyweight (represented as% initial body weight) (left) and colon length (right) of WT (n = 4) and a7HMZ (n = 4) mice treated as indicated. Significant comparisons are indicated with a P-value. (C) Left, representative H&E stain of WT and a7HMZ mice treated with 2.5% DSS for 5 days followed by 0 or 3 days of recovery. Right, histological scores of colitis in WTa7 (n = 4) and a7HMZ (n = 4) mice. (D) Left, IB for HNF4a (P1/P2 antibody) of WCE from the distal colon of a7HMZ mice treated as indicated. Right, representative IF of distal colon from a7HMZ mice treated with 2.5% DSS for 5 days -/+ recovery as indicated and stained with P1/P2-HNF4a antibody (green) and TO-PRO3 (red) for nuclei (40X magnification). Figure 4 continued on next page The isoforms share 76 interacting proteins, including previously reported HNF4g (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 HNF4a in a7HMZ and a1HMZ colons -138 and 99, respectively ( Figure 5C top and Figure 5C-source data 2A-E). Src tyrosine kinase, for example, bound uniquely in a1HMZ colons, consistent with our previous report that Src preferentially phosphorylates and interacts with HNF4a1 in cell-based and in vitro systems  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 a7HMZ 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 a7HMZ colons ( Figure 5B), and accelerated tumorigenesis in a7HMZ 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, the vast majority of which (62.9%) are known transcription regulators, protein kinases or phosphatases and associated proteins ( Figure 5C). For example, NDRG2, a kinase downstream of the mTOR/SGK pathway and a tumor suppressor that mediates apoptosis (Deuschle et al., 2012), and EMD, a nuclear membrane protein phosphorylated by Src (Tifft et al., 2009), both preferentially bind HNF4a1 and have been negatively associated with colon cancer. Likewise, HNF4a1 was preferentially bound by catalytic subunits of AMPK (PRKAA1/2) and is known to be phosphorylated by AMPK, which decreases its protein stability (Hong et al., 2003). However, AMPK suppresses cell proliferation via inhibition of mTOR and activation of p53 pathways (Motoshima et al., 2006) and low levels of AMPK activity are correlated with poor survival in metastatic colon cancer patients (Zulato et al., 2014), indicating that additional studies are required to elucidate the impact of AMPK signaling on HNF4a in colitis and colon cancer. In contrast, protein kinase C beta 2 (PRKCB) preferentially interacts with HNF4a7 and is known to be both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in the colonic epithelium (Liu, 2004). All told, there were hundreds of proteins that preferentially interacted with the HNF4a isoforms, including many signaling molecules as well as RNA binding proteins and transcription factors, providing multiple potential mechanisms for differential gene expression.

Differential effects on cell migration and chloride secretion in HNF4a isoform-specific mice
While the isoform-specific mice did not exhibit any overt morphological abnormalities in their intestines under normal conditions, the gene expression analysis (and AOM/DSS and DSS colitis results) suggested potential functional differences. Since there was a decrease in cell motility genes in a7HMZ mice, we examined migration of BrdU-labeled cells up the crypt and found that 48 hr after injection the average position of the BrdU-labeled cells (both in absolute terms and relative to the bottom of the crypt) was lower in a7HMZ mice compared to WT: this resulted in a statistically significant decreased migration of the BrdU+ cells during the 45 hr period ( Figure 5D and  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 a1HMZ (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 WTa1 (n = 8) and a1HMZ (n = 10) mice treated as in (E). The following figure supplements are available for Top, Venn diagram of total number of HNF4a-interacting proteins from RIME analysis found in a7HMZ only, a1HMZ only or both a7HMZ and a1HMZ 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 a7HMZ colons. Shown also are transcription factors that interact with HNF4a in both genotypes. Bold, proteins mentioned in text. Bottom, Total number of proteins in the indicated categories that show a significant interaction with HNF4a 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 HNF4a 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 Figure 5 continued on next page significantly higher in a1HMZ mice ( Figure 5D and Figure 5-figure supplement 1D,E). Despite these differences, there was a similar number of total BrdU+ cells in WT and a7HMZ mice (Figure 5-figure supplement 1F).
Since the ion transport genes were also decreased in a7HMZ, we examined electrogenic chloride secretion in isolated colonic mucosa. The distal colon of WT and a7HMZ mice exhibited a similar transmucosal electrical resistance and basal Isc (data not shown). However, the a7HMZ distal colon was refractory to stimulation with calcium-dependent (carbachol) and cAMP-dependent chloride secretagogues (forskolin), while the a1HMZ distal colon showed a markedly increased Isc response to forskolin ( Figure 5E). Since impaired chloride secretion is observed in colitis (Hirota and McKay, 2009), this differential response to forskolin as well as cell migration could explain, at least in part, the differential sensitivity of the a1HMZ and a7HMZ mice to DSS.

Elevated RELMb expression in a7HMZ mice plays a role in DSS sensitivity
During experimental colitis the cytokine RELMb is known to activate the innate immune system in response to loss of epithelial barrier function and increased exposure to gut microbiota: hence, mice lacking the Retnlb gene are known to be resistant to experimental colitis (Hogan et al., 2006;McVay et al., 2006). Interestingly, one of the genes most significantly upregulated in the untreated a7HMZ colon was Retnlb (5.3-fold increase versus WT controls); RELMb protein levels were also increased ( Figure 5F). In contrast, there was no significant difference in RELMb gene expression between a1HMZ mice and their WT littermates ( Figure 5F left).
To determine whether RELMb plays a causal role in the susceptibility of a7HMZ mice to colitis, we crossed a7HMZ mice with a RELMb knock out (Retnlb -/-) to generate RbKO/a7HMZ mice (Figure 6-figure supplement 1A). We confirmed that RELMb expression is lost in these mice, that HNF4a protein levels are unchanged by the RELMb knock out and that the a7HMZ allele has the same effect in the 'Rb line' (designated C57BL/6N+J, see Materials and methods for details) in terms of body weight loss and increased RELMb expression after DSS treatment ( Figure 6A and Figure 6figure supplement 1B-D).
Interestingly, while the histological score of the RbKO/a7HMZ mice was somewhat improved compared to WT/a7HMZ at three days of recovery, the difference was not significant (P=0.13) ( Figure 6B). In contrast, the weight loss of the RbKO/a7HMZ mice was completely restored to WT/ WT levels ( Figure 6C), as was the colon length ( Figure 6D) and overall survival ( Figure 6E). Notably, the RELMb protein levels in the a1HMZ mice were significantly reduced at three days of recovery (although elevated right at the end of the DSS treatment) and inversely correlated with colon length ( Figure 6F). These results suggest that elevated RELMb expression in untreated and DSS-treated a7HMZ mice and decreased expression during recovery in a1HMZ mice might contribute to their increased and decreased susceptibility to DSS-induced colitis, respectively. Interestingly, body weight loss is attenuated in a1HMZ during DSS treatment, however this protective effect is lost during recovery (Figure 6-figure supplement 1E). All together our results suggest that both P1-and P2-HNF4a are critical for full recovery following DSS treatment.  . RELMb knockout decreases susceptibility of a7HMZ mice to colitis. (A) RELMb protein level quantified by ELISA in the midcolon homogenate of mice with the indicated genotype treated with 2.5% DSS for 6 days. Genotypes are indicated as Retnlb/Hnf4a. N = 3-5 mice per genotype as indicated by each dot. (B) Histological scores of colitis in WT/WT (n = 6), WT/a7HMZ (n = 9) and Rb KO/ a7HMZ (n = 11) male mice treated with 2.5% DSS for 5 days followed by 3 days of recovery. Multiple sections per mouse were scored. (C) Percent change in body weight during and following DSS treatment (2.5% for 6 days). Day 0 is the start of treatment. Left, graph over time from one experiment. N = 3-5 mice per genotype. # Indicates P<0.05 on day 10 and 11 between WT/WT and a7HMZ/WT; * indicates P<0.01 on day 10 and 11 between a7HMZ/WT and a7HMZ/Rb KO. Right, meta-analysis of percent weight loss at 3 days of recovery after 6 days of treatment with 2.5% DSS from nine independent experiments. N = 12-47 mice per genotype. The WT/a7HMZ data include both the a7HMZ C57BL/6N parent as well as the a7HMZ C57BL/6N+J generated from the Retnlb-/-cross. See Figure 6-figure supplement 1 for a comparison of the two a7HMZ lines. (Data from one experiment in which all mice, including the WT/WT controls, had lower than normal body weight loss were excluded from the analysis.) (D) Colon length from mice treated with 2.5% DSS for 6 days followed by Figure 6 continued on next page

Direct and indirect mechanisms regulate RELMb expression in a7HMZ mice
To address the mechanism responsible for increased RELMb expression in a7HMZ mice we performed ChIP in CaCo2 cells, which express predominantly P2-HNF4a . We used the Support Vector Machine (SVM) learning tool in the HNF4 Binding Site Scanner to predict three potential HNF4a binding sites within 1.5 kb of the transcription start site (+1) of human RETNLB ( Figure 7A, left and Figure 7-figure supplement 1A-B), two of which are in the vicinity of NFkB and CDX2 binding sites (Wang, 2005;He et al., 2003). We found that endogenous HNF4a binds the two regions that encompass the SVM sites (Region 1 and 2) ( Figure 7A, right). The mouse Retnlb promoter also contains predicted HNF4a binding sites close to +1, one of which is highly conserved in human (Figure 7-figure supplement 1B-C, indicating that RELMb expression may be directly regulated by P2-HNF4a in both mouse and human. Luciferase assays in LS174T goblet-like cells with RELMb reporter constructs containing HNF4a binding sites in ChIP region 2 (Figure 7figure supplement 1D) confirmed that P2-HNF4a activates the RELMb promoter significantly more than P1-HNF4a ( Figure 7B). siRNA knockdown of endogenous P1-and P2-HNF4a in LS174T cells also showed a greater effect of loss of endogenous P2-HNF4a on RELMb expression than P1-HNF4a (Figure 7-figure supplement 1E). In contrast, on a known HNF4a-responsive promoter, ApoB, P1-HNF4a activates better than P2-HNF4a (Figure 7-figure supplement 1F).
Both P1 and P2-HNF4a are decreased after six days of DSS treatment in WT mice when RELMb expression is increased (Figures 2A and 6F), suggesting that additional mechanisms are at play in the upregulation of the Retnlb gene. Therefore, since RELMb expression is known to be activated by decreased epithelial barrier function (McVay et al., 2006), we conducted in vivo epithelial permeability assays using Fluorescein isothiocyanate-dextran 4 kDa (FITC-dextran) and found that a7HMZ mice have moderately decreased barrier function as shown by increased FITC-dextran in the serum of both untreated and DSS-treated mice ( Figure 7C). Furthermore, we found decreased FITC-dextran in a1HMZ mice compared to a7HMZ at 3 days of recovery ( Figure 7C), suggesting improved barrier function, consistent with the lower levels of RELMb and longer colon length in a1HMZ mice ( Figure 6F). Barrier function and colon length are both indicators of colon health.
Analysis of the expression profiling data in the untreated mice revealed dysregulation of several genes related to barrier function ( Figure 7D and Figure 5-source data 1G). For example, Il4i1 and Il13ra2, known signaling pathways critical for RELMb expression (Artis et al., 2004), are both increased in a7HMZ mice. There was also a concerted decrease in the expression of genes involved in cell adhesion and paracellular permeability in a7HMZ (Cdh1, Cldn15, Cldn16 and Sh3bp1), which would contribute to decreased barrier function and hence increased DSS sensitivity and RELMb expression in a7HMZ.

Discussion
The majority of mammalian genes have alternative promoters, which often result in different transcript variants, but relatively little is known about the physiological relevance of those transcript KO/a7HMZ (n = 9) mice after 6 days 2.5% DSS in one experiment. Meta-analysis of several independent experiments also showed that out of a total of 24 KO/a7HMZ mice allowed to go past 3 days of recovery, only one mouse died (3.6% mortality). In contrast, 13 out of 29 WT/a7HMZ mice (44.8%) either died or had to be sacrificed due to severe distress. Data for WT/a7HMZ mice in both the C57BL/6N and C57BL/6N+J lines were combined: no difference in mortality was noted between the lines. (F) WT, a7HMZ and a1HMZ mice (all in C57BL/6N background, n = 3-63-6 per genotype per treatment) were treated with 2.5% DSS for 6 days alone or followed by 3 days of recovery. Left, RELMb protein quantified by ELISA in the midcolon homogenate: shown are means of technical triplicates from one experiment. Right, colon length. RELMb ELISA: *P<0.03 versus WT; # P<0.01 versus a7HMZ. Colon length: *P<0.01 versus WT; # P<0.0002 versus a7HMZ. The following figure supplement is available for Figure 6 variants (Djebali et al., 2012;Xin et al., 2008). Here, we examined the HNF4a gene, which has two highly conserved promoters (P1 and P2) that give rise to proteins with different N-termini (P1-HNF4a and P2-HNF4a). Even though both promoters are expressed in adult intestines (Tanaka et al., 2006;Nakhei et al., 1998;Brianç on and Weiss, 2006) and HNF4a has been implicated in human colon cancer Tanaka et al., 2006;Oshima et al., 2007;Cancer Genome Atlas Network, 2012;Zhang et al., 2014) and colitis (Ahn et al., 2008;Barrett et al., 2009;Fang et al., 2011), the distribution and function of the different HNF4a isoforms in the colon have not been investigated until now (results summarized in Figure 7E). We show that in untreated WT adult mice P1-HNF4a protein is expressed in the differentiated compartment and the surface of the colonic epithelium whereas P2-HNF4a expression is restricted to the proliferative and differentiated compartments ( Figure 7F). Transgenic mice expressing just a single isoform of HNF4a developed morphologically normal intestines, but hundreds of genes in the colon were differentially expressed compared to WT mice, many of which are consistent with altered barrier function and localization of the isoforms: mice expressing only P1-HNF4a (a1HMZ) had an upregulation of genes involved in differentiation, while mice expressing only P2-HNF4a (a7HMZ) had higher levels of DNA repair and cell cycle genes. Furthermore, we found that HNF4a isoforms interact in vivo with unique sets of proteins, especially those involved in signal transduction, which could contribute to differential gene expression patterns and thereby result in different susceptibilities to colitis and colitis-associated colon cancer in the isoform-specific mice. Finally, we show that Retnlb, which encodes the cytokine RELMb, a key player in DSS-induced colitis, is a downstream target of P2-HNF4a.

Role of HNF4a isoforms in colon cancer
In a mouse model of CAC, a1HMZ mice exhibited decreased tumor load, suggesting that expression of HNF4a1 from the P2 promoter in the proliferative compartment may protect against tumorigenesis, consistent with studies showing a loss of P1-HNF4a in human colon cancer Tanaka et al., 2006;Oshima et al., 2007), a tumor suppressive role for P1-HNF4a in mouse liver (Hatziapostolou et al., 2011;Walesky et al., 2013b) and our recent colon cancer xenograft studies showing that ectopic expression of P1-HNF4a reduces tumor growth (Vuong et al., 2015). In contrast, a7HMZ mice showed an initial increase in tumorigenesis, which could reflect the absence of P1-HNF4a. Since there was no increase in the number of Ki67-or BrdU-positive cells in a7HMZ colons, no visible tumors after a chronic colitis regimen (three cycles of DSS treatment) (data not shown) nor acceleration of tumor growth in the presence of ectopic expression of P2-HNF4a in the xenograft model (Vuong et al., 2015), there is no indication that P2-HNF4a actively promotes proliferation. Rather, P2-HNF4a appears to be merely permissive of cell proliferation, consistent with its expression in the proliferative compartment and its retention in human colon cancer Tanaka et al., 2006).. Interestingly, HNF4a has been shown to act as an Figure 7. Direct and indirect mechanisms of regulation of RELMb expression by HNF4a isoforms: impact on DSS sensitivity and recovery. (A) P2-HNF4a binds the promoter of the RETNLB gene in colonic epithelial cells. Left, schematic of the human RETNLB promoter showing predicted SVM binding sites for HNF4a, as well as sites for NFkB, KLF4, STAT6 and CDX2 (He et al., 2003). Right, ChIP results for endogenous HNF4a in Caco2 cells at RELMb Region 1 and Region 2, as well as an HMOX1 control. In, input (1/10 dilution); Ig, IgG; H4, anti-HNF4a. (B) Left, uciferase activity of pGL2.basic and RELMb reporter constructs in LS174T cells cotransfected with vector (pcDNA3.1), human HNF4a2 or HNF4a8. Shown is the RLU normalized to protein concentration. Data are represented as mean of triplicates + SD of one independent experiment. *P<0.05, **P<0.005 between vector control and HNF4a2 or HNF4a8. $ P<0.05, $$ P<0.005 HNF4a2 versus HNF4a8. Right, IB of extracts from LS174T cell line cotransfected with -870.hRELMb reporter and HNF4a isoforms. (C) Gut permeability measured by appearance of FITC dextran (4 kDa) in serum of WT, a7HMZ and a1HMZ mice either untreated, treated with 2.5% DSS for 6 days alone or followed by 3 days of recovery. (n = 7-10 for all groups except a7HMZ 6d DSS + 3d recovery where n = 4). P-values were determined by Student's T-test. (D) List of genes related to barrier function altered in the distal colon of a7HMZ and a1HMZ mice compared to their WT controls. Shown is nonlog fold change from the microarray experiment in Figure 5. (E) Summary of various phenotypes of HNFa isoform-specific mice (a7HMZ and a1HMZ) relative to WT mice in untreated, DSS and AOM+DSS treated animals as indicated. n. oncogene in gastric cancer and only P2-HNF4a is expressed in the stomach (Chang et al., 2014;Dean et al., 2010).

Role of HNF4a isoforms in DSS-induced colitis
An additional and/or alternative explanation for the differences in CAC-induced tumors in the isoform-specific mice could be their remarkable differences in DSS sensitivity, which in turn could be due to opposing chloride secretory responses and epithelial migration ( Figure 7E). Since the chloride secretory pathway is required for maintaining proper luminal hydration, which helps protect the epithelium from physical damage (Barrett and Keely, 2000), this suggests that the isoform-specific mice have different barrier functions, which we confirmed with FITC-dextran assays. Decreased expression of cell adhesion genes in a7HMZ colons -such as E-cadherin (Cdh1), an established HNF4a target Elbediwy et al., 2012) critical for both migration of cells along crypt-villi axis and epithelial barrier function (Grill et al., 2015;Schneider et al., 2010) -would contribute to decreased barrier function. In contrast, downstream effectors of IL-18 signaling (Il18r1 and Il18rap), which are implicated in intestinal epithelial barrier function (Nowarski et al., 2015), were increased in a7HMZ mice, while Il18bp, a decoy receptor for IL-18 which attenuates signaling, is decreased. All told, there are several key cell adhesion and cytokine signaling genes that are dysregulated in a7HMZ colons that could contribute to decreased barrier function and subsequently a pro-inflammatory state (Hogan et al., 2006;McVay et al., 2006), which could in turn contribute to the enhanced colitis and tumorigenesis observed in a7HMZ mice. One such cytokine is RELMb, which we show is a direct target of HNF4a and preferentially activated by P2-HNF4a.
DSS also causes epithelial injury and a need for rapid proliferation, expansion, migration and differentiation of intestinal epithelial cells to promote wound healing and regeneration (Sturm, 2008). Hence, the inability of a7HMZ mice to effectively recover from DSS could be attributed to defective migration, chloride secretion (ion transport) and/or differentiation ( Figure 7F). BrdU-labeled cells exhibited greater migration in a1HMZ and lower migration in a7HMZ mice: a7HMZ mice also had reduced expression of genes involved in cell motility. Cldn15 is downregulated in a7HMZ and upregulated in a1HMZ colons and a known target of HNF4a (Darsigny et al., 2009). Cldn15 dysregulation could explain the decrease in secretory capacity in a7HMZ mice and hence their inability to recover after DSS as a basal level of secretion is important for proper gut formation (Anderson and Van Itallie, 2009;Tamura et al., 2008). Cdx2, another established target of HNF4a (Saandi et al., 2013) and a major player in intestinal differentiation (Suh and Traber, 1996;Lorentz et al., 1997), has a similar expression profile. Finally, the involvement of these processes in recovery from DSS could explain why the crypt structure in the a7HMZ mice is not completely ameliorated by the RELMb knockout even though body weight and colon length loss and lethality are: it has been noted previously that RELMb expression per se does not alter colonic epithelial proliferation McVay et al., 2006 and its role in affecting the barrier function is still debated (Hogan et al., 2006;McVay et al., 2006).
In summary, the results presented here indicate that while P1-and P2-HNF4a isoforms can substitute for each other during normal development and homeostasis, under conditions of stress they play notably different roles. Those roles seem to be driven by unique interacting partners leading to differential expression of target genes. The results also show that any factor that disrupts the balance between the HNF4a isoforms in the colon could have serious functional consequences. Those factors include Src tyrosine kinase , as well as any one of a number of other signaling molecules that interact preferentially with the isoforms. Future studies will be required to elucidate all the underlying mechanisms but it is anticipated that several will be important in diagnosing and treating gastrointestinal diseases involving HNF4a.

Animal use and care
Care and treatment of animals were in strict accordance with guidelines from the University of California Riverside Institutional Animal Care and Use Committee (Protocol number A200140014). Mice were maintained in isolator cages under 12 hr light/dark cycles at~21˚C on bedding (Andersons bed OCOB Lab 1/8 1.25CF) from Newco (Rancho Cucamonga, CA) and either fed a standard lab chow (LabDiet,#5001,St. Louis,MO) and maintained in an open access vivarium or fed an irradiated chow (LabDiet, #5053) and housed in a specific pathogen-free (SPF) vivarium (a7HMZ and Retnlb -/matings). All experiments were performed in an open access vivarium except those in Figure 4CEF where the mice were born in an open-access vivarium and then moved to an SPF facility before treatment (due to a required institutional change). Subsequently, mice born in the SPF facility were brought to an open access facility at least two weeks prior to DSS treatment.
Transgenic mice on a mixed 129/Sv plus C57BL/6 background carrying exon 1A or exon 1D in both the P1 and P2 promoter (a1HMZ or a7HMZ, respectively) have been described previously (Brianç on and Weiss, 2006). Both lines were maintained as heterozygotes (HTZ); wildtype (WT) and homozygous (HMZ) were mated for a single generation to generate mice for experiments. Appropriate, age-matched WT controls for both the a1HMZ and a7HMZ lines were used (designated WTa1 and WTa7, respectively, Figures 1-5). The a7HMZ and a1HMZ mice were backcrossed to C57BL/ 6N for 10+ generations and used with C57BL/6N WT controls ( Figure 6). The backcrossed a7HMZ mice were crossed with RELMb knockout (Retnlb -/-) mice which were generated as previously described (Hogan et al., 2006) using VelociGene technology. The Retnlb -/mice were backcrossed 6+ generations in C57BL/6J to generate RbKO/a7HMZ mice in a C57BL/6N+J background. WT/ a7HMZ mice from the RELMb cross showed essentially identical susceptibility to DSS as the a7HMZ parent in the C57BL/6N background (as well as the original exon swap mice in the mixed background) ( Figure 6-figure supplement 1C and data not shown). All experiments with RbKO/a7HMZ mice were compared to RbWT/a7HMZ from the RELMb cross except for the meta analysis in Figure 6C which included data from the parental a7HMZ line in C57BL/6N. Mice of the same genotype were housed three to five per cage, randomly assigned to treatment groups at the beginning of the experiment and subjected to a single experimental regime in their cages. Mice were euthanized by CO 2 asphyxiation and tissues harvested in the mid morning to mid afternoon.

DSS colitis
Male mice (10 to 16 weeks old) were treated with 2.5% DSS (MW 36,000-50,000 Da, MP Biomedicals, #160110, Santa Ana, CA) in water given ad libitum for four to six days and sacrificed immediately or allowed to recover up to 18 days with tap water. WT mice were treated in parallel in each experiment as controls for the DSS: the same lot number of DSS was used for a given group of experiments whenever possible to avoid lot-to-lot variation. Mice in severe distress (weighing 13 grams or less, or excessively hunched and lethargic) were euthanized prior to the termination of the experiment except in experiments measuring mortality. To avoid confounding effects due to unrelated illnesses, when an animal became unexpectedly ill, all mice in the cage were excluded from the analysis.
Colitis-associated and sporadic colon cancer CAC was established as described (Neufert et al., 2007). Briefly, we intraperitoneally (i.p.) injected male mice (6 to 10 weeks old) with 10 mg/kg AOM (National Cancer Institute, Bethesda, MD) on Day 1 in the morning. On Day 2 mice were given 2.5% DSS in water for four to seven days, followed by 16 days of untreated water; the cycle was repeated one or two additional times. Mice were sacrificed at day 46 to 95; tumor number counted by visual inspection and tumor size measured with digital calipers were determined in a blind fashion. Sporadic colon cancer was induced in male mice (6 to 8 weeks old) by i.p. injection of 10 mg/kg AOM once a week for six consecutive weeks. Mice were sacrificed 28 weeks after the first injection.

BrdU labeling
Young adult male mice were injected i.p. with 75 mg/kg BrdU (BD Biosciences, #550891, San Jose, CA) and sacrificed after 2 to 3 hr, 25 hr or 48-50 hr. Distal colons fixed in formalin were sectioned and immunostained with BrdU antibody as per manufacturer instruction (BD Biosciences, #550803). Images were captured at 40X (Zeiss Axioplan, Jen Germany) and crypt dimensions were measured using SPOT Imaging software (Sterling Heights, MI).

Isolation of mouse colonic crypts
Distal colons from 19-week-old male WT mice from the mixed background (WTa7) were rinsed in PBS, placed in a 4% bleach solution for 20 min, washed three times in PBS and then incubated with 3 mM EDTA, 0.5 mM (DTT) in PBS for 90 min at 4˚C followed by a PBS wash. Colonic crypts were isolated by vigorous shaking; they were fixed and immunostained as described .

Ussing chamber assay
The short circuit current (I sc ) and electrical resistance across the mucosal layer of mouse distal colon was measured using an Ussing chamber as described (Bajwa et al., 2009). Electrogenic Cl À secretion was recorded as the I sc evoked by sequential addition of 100 mM carbachol and 20 mM forskolin (Sigma-Aldrich) to the serosal bath.

Expression profiling
Mouse Exon 1.0 ST Arrays (Affymetrix, Santa Clara, CA) were hybridized at the University of California Riverside Genomics Core using polyA+ RNA extracted from the distal colon of young adult male mice (12 to 16 weeks old) fed a standard lab chow ad libitum in an open access vivarium. RNA was pooled from two to three mice per genotype and applied to one array; a second array was processed in a similar fashion for a total of four to six mice assayed per genotype. Results from the two arrays were averaged. Isoform-specific mice were compared to their appropriate, age-matched WT controls. Data were analyzed using Robust Multi-array Average (RMA) background adjustment and quantile normalization on probe-level data sets with Bioconductor packages, Exonmap, and Affy software. To determine the differentially expressed transcripts only the probes with P<0.05 (Student's t-test) and more than two-fold change were considered. Gene Ontology (GO) overrepresentation analysis was conducted using DAVID. Microarray data have been deposited in the Gene Expression Omnibus MIAME-compliant database (Accession number GSE47731) (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE47731).

RELMb ELISA
Colon tissue (~1 cm) was weighed and homogenized in 0.5 mL PBS, followed by ELISA with capture and detection biotinylated antibodies for anti-RELMb (Cat #500-P215Bt, Peprotech) according to the manufacturer's instructions. Samples were compared to a serial-fold dilution of recombinant mouse RELMb protein (#450-26B, Peprotech) and calculated as ng per gram tissue. All ELISAs were performed in technical triplicates.

Fluorescein isothiocyanate-dextran (FITC-dextran) assay
Intestinal epithelial permeability was assessed by measuring the appearance of FITC-dextran (FD-4, Sigma) in mouse serum as described previously (Brandl et al., 2009). Untreated or treated (2.5% DSS for six days or 2.5% DSS for six days, followed by days of recovery with tap water) WT, a7HMZ or a1HMZ mice were fasted overnight and then gavaged with FITC-dextran (60 mg/100 g body weight) 4 hr before harvesting. Blood was collected either from the inferior vena cava or by cardiac puncture and allowed to sit on ice for 30 min. Serum was collected after centrifuging the blood for 15 min at 2000 xg at 4˚C. FITC-dextran measurements were performed in duplicate or triplicate by fluorometry at 490 nm. Data were analyzed using Prism 6 software (GraphPad Prism version 6 for Mac, GraphPad Software, La Jolla, CA); outliers were identified by the ROUT method and removed.
ON-TARGET siRNA targeting P1-and P2-HNF4a were custom synthesized from Dharmacon. si P1-HNF4a: Sense, 5'-U U G A G A A U G U G C A G G U G U U U U -3'; Antisense 3'-U U A A C U C U U A C A C G U C C A C A A -(5'-P) 5'. siP2-HNF4a: Sense, 5'-G U G G A G A G U U C U U A C G A C A U U-3'; Antisense, 3'-U U C A C C U C U C A A G A A U G C U G U-(5'-P) 5'. ON-TARGETplus Non-targeting siRNA #1 (D-001810-01-20) was used as siControl.
Proteins had to be present in at least two out of the three replicates with the HNF4a antibody and not in any IgG control in both a1HMZ and a7HMZ samples to be considered for the 'both' category. To be considered in the 'a7HMZ only' category, the protein had to appear either in two of three a7HMZ samples but in none of the three a1HMZ samples, or in three of the a7HMZ samples and only one of the a1HMZ samples. A similar strategy was used for the 'a1HMZ only' proteins. Proteins were converted to gene symbols and cross-referenced with human and mouse genes associated with colon cancer, IBD, Crohn's disease and ulcerative colitis found in the literature (Franke et al., 2010;Jostins et al., 2012) and a Pubmed-Gene search conducted in April 2016, followed by manual curation. Gene Ontology using Panther (www.pantherdb.org) as well as manual curation resulted in the TF, RNA binding and protein kinase and phosphatase categories in Figure 5C bottom. The Human Protein Atlas (http://www.proteinatlas.org/) was used to confirm expression in the colon and nucleus.

Statistical analyses
Sample sizes for DSS and AOM+DSS regimes were determined on the basis of mouse-to-mouse variation in body weight loss and tumor number/load (respectively) observed in pilot experiments. Each mouse was considered to be a biological replicate; technical replicates refer to multiple analyses of the same tissue from a given animal. All results are expressed as the mean ± s.e.m, of sample size n. Significance was tested by analysis of variance or Student's t-test. Probabilities less than 5% (P<0.05) were considered to be significant. For RIME, a cut off of q<0.01 (1% FDR) was used.