TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors

Abstract

SummaryUnderstanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

5-Hydroxymethylcytosine (5hmC), or How to Identify Your Favorite Cell

“…In adult livers, such 5hmC occupancy is overrepresented at genes involved in active catabolic and metabolic processes [71] (Figure 3). While these studies suggest a role of 5hmC in liver physiology, a recent report described a specific 5hmC event during hepatocyte differentiation [72]. TET-dependent 5hmC activation preceded activation of the HNF4A P1 promoter, a master TF of hepatocyte identity (Figure 3).…”

“…Recent studies are shedding light into such mechanisms. For example, interaction between TET proteins and lineage-specific TFs such as the FOXA family have been described [72,89,100]. Although this is better supported at the level of pioneer factors, it is feasible that other TFs involved in lineage commitment are also able to recruit TETs to specific genomic loci.…”

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro hepatocyte differentiation

“…HepaRG cells are bipotent liver progenitor cells that differentiate in vitro after 4 weeks into either hepatocytes or cholangiocytes. Our group previously found a TET1-dependent switch from methylated to hydroxymetylated DNA status at HNF4A promoter P1 in HepaRG cells, triggering differentiation at one week of cell culture [Ancey et al, 2017]. In order to determine the gene expression profile at this stage of hepatocyte differentiation ( Figure 1A), RNA was isolated and a transcriptome analysis was performed to identify differentially expressed genes (DEGs) ( Figure 1B).…”

“…Considering that at 1 week of HepaRG differentiation there is a TET1-mediated 5hmC enrichment on HNF4A promoter P1 [Ancey et al, 2017] and the transcriptome already reflects a hepatocyte-like profile, we chose this time point to assess 5hmC levels of the HepaRG cell line compared to its proliferative state. Immunostaining against 5hmC reveals the presence of this modified cytosine in differentiating cells, in contrast with its almost complete absence in proliferative HepaRG cells (Figure 2A and Supplementary Figure S2).…”

“…Moreover, 5hmC is emerging as a stable modified base with unique functions that suggests that the dynamic distribution of 5hmC could be an acquired "imprint" of cell identity during adult progenitor cell differentiation in several tissues (reviewed in [Ecsedi et al, 2018]) including liver. Indeed, using an in vitro model of hepatocyte differentiation, we recently described a switch in 5mC/5hmC state of promoter 1 (P1) of the master transcription factor (TF) of hepatocyte identity HNF4A [Ancey et al, 2017].…”