Cellular proliferation and differentiation are critical events in normal development and cancer. Despite decades of research, much remains to be learned about how cells transition between the two states. To decipher one aspect of this switch, we focused on the transcription factor, Hepatocyte Nuclear Factor (HNF) 4 alpha. HNF4a is a nuclear receptor that is important in development and in maintaining the homoeostasis of the adult liver and colon. There are multiple isoforms of the HNF4a that are generated by alterative promoter (P1 and P2) usage and 3' splicing events in different tissues. Both P1 and P2-HNF4a isoforms are expressed in the adult colon, while the P1-HNF4a is expressed in the adult liver. Others have shown that P1-HNF4a is downregulated in cancer, whereas P2-HNF4a is upregulated in hepatocellular carcinoma and colorectal cancer. This would suggest that P1-HNF4a is tumor suppressive, while P2-HNF4a may act as an oncogene although a mechanism has not been elucidated. One potential mechanism could be through a differential interplay with the Wnt/beta-catenin/TCF pathway, which is activated in many cancers including liver and colon. To determine whether HNF4a and TCF4 cross-talk to control the switch between proliferation and differentiation and to elucidate the role of the HNF4a isoforms in early development and cancer, we generated Tet-On inducible systems that express either HNF4a2 (P1) or HNF4a8 (P2) under the control of doxycycline in human colon cancer and mouse embryonic stem cells. We characterized the lines to look for morphological and functional differences between the isoforms. We performed RNA-Seq and ChIP-Seq on the HCT116 inducible lines to identify any changes in gene expression and regulation and compared the HNF4a2 and HNF4a8 lines to determine functional differences. Although we found some functional redundancy, there are discrete differences. HNF4a2 suppresses tumor growth, inhibits cellular proliferation, and competes with TCF4 for regulation of target genes more effectively than HNF4a8. Similarly, we also found that HNF4a2 decreases cell numbers more than HNF4a8 in the mES inducible lines. Our findings provide insight into the distinct role of the P1- and P2-HNF4a in cancer and normal development.

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.