Genome-wide changes in chromatin accessibility in myoblasts lacking Daxx [ATAC-Seq]

DAXX, a H3.3 histone chaperone known for its role in heterochromatin maintenance, has been understudied in the context of gene expression regulation. In this study, we generated Daxx knockout myogenic cells and observed a significant loss of myogenic markers expression and impaired differentiation. Transcriptome analysis revealed broad dysregulations in Daxx KO cells, including loss of myogenic identity and a concurrent upregulation of genes involved in DNA replication and telomere maintenance. Chromatin immunoprecipitation followed by sequencing demonstrated a marked reduction in H3.3 deposition, particularly in intronic and intergenic regions. Further analysis indicated that loss of DAXX leads to decreased H3K27ac at myogenic loci and a shift in repressive histone marks, which led to impaired gene expression. Intriguingly, double knockout of Daxx and Hira resulted in distinct transcriptomic alterations, demonstrating that DAXX and HIRA have both overlapping and unique roles in H3.3 incorporation. Our work also suggests the presence of additional histone chaperone complexes in maintaining chromatin integrity in myoblasts. Our findings establish DAXX as a critical regulator of myogenic gene expression and muscle cell identity through a distinct mechanism from that of HIRA and highlighted an unanticipated plasticity in the deposition loci for DAXX and HIRA in myoblasts. Overall design: Examining the changes in H3.3 incorporation and associated changes in histone modifications in the absence of Daxx in myoblasts

DAXX（一种以参与异染色质维持功能而闻名的H3.3组蛋白伴侣（H3.3 histone chaperone））在基因表达调控领域的研究仍较为匮乏。本研究构建了Daxx基因敲除的成肌细胞（myoblasts），观察到成肌标志物的表达显著降低，且细胞分化能力受损。转录组分析（Transcriptome analysis）显示，Daxx敲除细胞存在广泛的表达失调：不仅丧失了成肌细胞身份特征，同时上调了一系列参与DNA复制与端粒维持的基因。染色质免疫共沉淀测序（Chromatin immunoprecipitation followed by sequencing）结果表明，H3.3的沉积水平显著降低，尤其在内含子及基因间区域。进一步分析发现，DAXX缺失会导致成肌基因位点的H3K27乙酰化修饰（H3K27ac）水平下降，并改变抑制性组蛋白修饰谱，最终造成基因表达异常。值得注意的是，Daxx与HIRA的双基因敲除会引发独特的转录组改变，这表明DAXX和HIRA在H3.3组蛋白掺入过程中既存在功能重叠，也具备各自独有的作用。本研究还提示，成肌细胞中可能存在其他组蛋白伴侣复合物以维持染色质完整性。我们的研究结果证实，DAXX是调控成肌基因表达与肌细胞身份的关键因子，其作用机制不同于HIRA；同时也揭示了成肌细胞中DAXX与HIRA介导的H3.3沉积位点存在未被预期的可塑性。实验整体设计：探究成肌细胞中Daxx缺失后，H3.3组蛋白掺入水平的变化及其伴随的组蛋白修饰改变。