Roles of H2A.Z histone variant isoforms during enterocyte-like differentiation process (RNA-Seq)
The involvement of the histone variant H2A.Z and its isoforms in the regulation of gene expression is an increasing exciting field considering the impact of such regulations in physio-pathology. Indeed, we and other recently showed that H2A.Z.1 and H2A.Z.2 isoforms exert cooperative or antagonistic transcriptional regulations on subsets of genes involved in key processes, such as proliferation, senescence or several organ functions. In this work, we analyze the relative role of both H2A.Z isoforms on parameters of the intestinal epithelial homeostasis. We observed that the amount of H2A.Z.1 and H2A.Z.2 at TSS and gene bodies are highly correlated and that the two H2A.Z isoforms can replace each other when depleted. We highlighted the role of their respective deposition onto chromatin by specific incorporators in some discrete isoform-specific contribution to the differentiation process. We have also uncovered an unexpected link between H2A.Z isoforms occupancy at gene bodies and the propensity of genes to be induced in enterocyte differentiation. Overall design: Caco-2/15 cells is a useful model to study the intestinal enterocyte-type differentiation process, since it spontaneously acquired lineage-specific differentiation features upon confluence (Van Beers et al., 1995). To decipher the contribution of each H2A.Z isoform to gene expression pattern modifications that occur during intestinal epithelial differentiation, we first generated Caco-2/15 cell -based models in which we were able to heterozygously introduce a Flag tag into the 3' regions of H2AFZ or H2AFV genes, by CRISPR-Cas9 genome editing, to bypass the lack of isoform-specific antibody. These models allowed us to follow, during the differentiation process, patterns of each isoform expression and incorporation into chromatin.  Using these models, we performed ChIP-seq and RNA-seq experiments for cell cultures in subconfluent (Sc) or 7-days post-confluent (Pc) state, allowing us to highlight H2A.Z isoforms enrichment and regulated genes subsets during differentiation.