H2AZ extended acidic patch is necessary for formation specialized chromatin states in ESCs [ChIP-Seq]. Mus musculus

The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation. Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ –depleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification. Overall design: H2AZ extended acidic patch was mutated, or H2AZ was KD in mouse embryonic stem cells and RNA-Seq analysis was performed on the resulting cultures. Characterization of H2AZ-WT and -AP3-mutant binding specificities were performed by ChIP-Seq.

组蛋白H2A变体H2AZ对于胚胎发育，以及胚胎干细胞（embryonic stem cells, ESCs）中发育相关基因表达程序的正常执行至关重要。H2AZ的可变区域可能是其功能特化的关键所在，但目前我们对这些序列差异如何参与染色质调控的机制知之甚少。
本研究证实，延伸型酸性结构域（extended acidic patch, AP）——尤其是C端对接结构域中的三个可变残基——对于胚胎干细胞分化过程中的细胞谱系定型，以及分化阶段基因表达程序的正常执行必不可少。令人意外的是，与H2AZ敲低（knockdown, KD）相比，破坏酸性结构域会对细胞特性塑造产生截然不同的影响。这与早期细胞谱系定型阶段，H2AZ敲低胚胎干细胞与酸性结构域（AP）突变胚胎干细胞的基因表达谱差异相一致。
有趣的是，酸性结构域突变体的表达对基因调控产生的独特影响，与染色质状态发生改变且变得不稳定、同时依赖于活跃转录的高染色质流动性现象相吻合。综合来看，本研究数据表明，酸性结构域内的可变残基是H2AZ功能发挥的关键结构决定因素，并将染色质结构与动态变化同基因调控及细胞命运定型联系起来。
实验设计方案：在小鼠胚胎干细胞中对H2AZ的延伸型酸性结构域进行突变，或对H2AZ进行敲低（KD），随后对培养细胞开展RNA测序（RNA-Seq）分析；通过染色质免疫共沉淀测序（ChIP-Seq）对H2AZ野生型（H2AZ-WT）与AP3突变体（H2AZ-AP3-mutant）的结合特异性进行表征。