| Developmental biology studies have traditionally been performed using animal models that house evolutionarily conserved pathways relevant to human biology. Although animal model systems, including mice, rats, zebrafish, chick and xenopus, have allowed for great advance in the understanding of molecular mechanisms controlling hepatic development, we aimed at using a human system to model human development. Therefore, we sought to use pluripotent stem cells and their differentiation into hepatocyte-like cells as a model to study the role of HNF4a during human hepatocyte development. The hepatocyte differentiation protocol has been shown to mimic key stages during hepatocyte differentiation, including definitive endoderm, specified hepatic cells, immature hepatocytes and mature hepatocytes. Each stage is defined by the expression of key markers that are present at that developmental time point during liver development in vivo..;HNF4a is a nuclear hormone receptor that is critical during hepatocyte development, and has been shown to be essential for differentiation of hepatocytes, as well as hepatocyte function in mice. Although its role during liver development has been well studied, how it functions to control transcription of its target genes is still unknown. HNF4a has been shown to interact with many co-factors, including CBP/p300, SRC1, GRIP1 and PRMT1. These enzymes induce both histone acetylation as well as arginine methylation at distinct amino acids on histones. We hypothesized that HNF4a controls hepatocyte differentiation by recruiting co-factors to the promoters of liver specific genes, to induce acetylation of surrounding histones that repositions nucleosomes and allows for recruitment of RNA Pol II to the transcriptional start site to activate transcription.;To determine if HNF4a is required for proper human hepatocyte differentiation we used a lenti-viral shRNA system to knock down HNF4a in human pluripotent stem cells. Upon differentiation of these cells into hepatocytes, we found that HNF4a expression is lost as compared to wild type cells. We saw a loss of hepatocyte markers at the mature hepatocyte stage in the HNF4a depleted cell line. From these data we concluded that HNF4a is required for the proper differentiation of human hepatocytes. To narrow down the stage of differentiation in which HNF4a is first required, we defined a global mRNA fingerprint at the transition of endoderm into hepatic progenitor cells. Upon the loss of HNF4, we saw a decrease in expression of several hepatic mRNAs three days after the addition of BMP4 and FGF2 to pluripotent cell derived endoderm. From this work we concluded that HNF4a is required for human hepatic specification by controlling expression of genes characteristic of the hepatic fate.;Next, we identified HNF4a direct targets during hepatic specification by ChIP-seq. To identify binding sites that are functionally relevant during hepatic specification, we crossreferenced our ChIP-seq data with global expression profiles, with and without HNF4a. We identified 11 candidate genes where expression is lost when HNF4a levels are depleted, the HNF4a binding site lies within 5 Kb upstream of its TSS, and the binding of HNF4a is reproducible between experiments. We predicted that HNF4a was recruiting co-factors to its target genes to alter chromatin state, therefore, we examined chromatin modifications at several of the HNF4a target genes, with and without HNF4a. Data from ChIP experiments with antibodies against histone modifications three days after the addition of BMP4 and FGF2 to endoderm show that upon loss of HNF4a we saw a decrease in RNA Pol II presence at HNF4a targets as compared to control. Additionally, we saw a decrease in a histone modification most commonly found at active enhancers, H3K27Ac. HNF4a has previously been shown to interact with CBP/p300, the enzyme that acetylates lysine 27 on histone H3. From these data we can conclude that HNF4a is the factor responsible for H3K27ac at its target genes, as well as the recruitment of RNA Pol II. We propose a model for the molecular function of HNF4a during the differentiation of human hepatocytes. We predict that HNF4a binds to its consensus site at hepatic genes, recruits CBP/p300 to acetylate H3K27. Acetylation of H3K27 alters the chromatin environment to make it amenable for RNA Pol II to bind and actively transcribe HNF4a target genes.;In summary, we have used the differentiation of pluripotent stem cells into hepatocytes to define the developmental time point in which HNF4a is required. We have also used this model system to show that HNF4a is the key molecule in the recruitment of RNA Pol II at its target genes during the differentiation of endoderm into hepatic progenitor cells. |
