Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

Abstract

During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4a) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4a is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4a 0 s Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4a during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation.

Multi-omics Analysis of the Intermittent Fasting Response in Mice Identifies an Unexpected Role for HNF4α

“…From these images, we noted that the HNF4a western blot showed the presence of 2 bands in mouse liver extracts. The lower band corresponded to the predicted molecular weight of mouse HNF4a ($53 kDa), and the upper band corresponded to SUMO2-modified HNF4a described recently in human hepatocytes (Wang et al, 2019;Zhou et al, 2012). Statistical analysis of each of these bands between the four treatment groups showed that the lower band was significantly increased only by acute fasting, consistent with our proteomics dataset, but the upper band showed a statistically significant decrease in livers of animals harvested in the fasted state from either diet group ( Figure 6C).…”

Highlight report: role of HNF4α in stem-cell differentiation to hepatocytes

“…One common problem is too low expression of hepatocyte-specific genes; a further challenge is expression of 'unwanted genes' that are not observed in primary human hepatocytes (Sachinidis et al 2019;Godoy et al 2015). The present systematic study of Wang et al (2019) showed that HNF4α does not only play a role in the steps of final differentiation from hepatoblasts to mature hepatocytes but also during development from endoderm to hepatoblasts.…”

“…It contains several functional regions, the N-terminal transactivation domain (AD-1), a zinc finger DNA-binding domain, a flexible hinge region, a multifunctional ligand-binding domain with a second transactivation domain (AD-2), and a repression region (Lau et al 2018;Dhe-Paganon et al 2002). In the present study, Wang et al used CRISPR-Cas 9 genome-editing techniques to understand the role of the DNA-binding domain and the transactivation domains (AD-2) during differentiation of stem cells to hepatocyte-like cells (Wang et al 2019). Interestingly, the two modifications had a different influence on stem-cell specification.…”

“…Interestingly, the two modifications had a different influence on stem-cell specification. Truncation of the DNA-binding domain blocked differentiation even to hepatic progenitor cells; in contrast, modification at AD-2 still allowed differentiation of stem cells to hepatocyte progenitors, but further expression of genes characteristic for mature hepatocytes was compromised (Wang et al 2019).…”

“…Recently, Wang and colleagues from Edinburgh University performed a study, where they generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to study their influence during differentiation to hepatocyte-like cells (Wang et al 2019). HNF4α is a transcription factor that plays a major role for gene expression in hepatocytes (Sladek et al 1990).…”