Hmga2 protein loss alters nuclear envelope and affects 3D chromatin structure upon the induction of pluripotent stem cell commitment [HiC]

Hmga2 KO pluripotent stem cells fail to develop into epiblast-like stem cells (EpiLCs). By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in stabilized epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. As nuclear lamina is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of HiC data in wt and Hmga2 KO cells allowed us to observe that inter-TAD interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. Comparative gene expression profiling analysis of RNA-seq data for Hmga2 wt and KO PSCs induced to differentiate into EpiLCs. Genome binding/occupancy profiling of Hmga2 in EpiLCs by high throughput sequencing. High-throughput chromatin conformation capture sequencing (HiC-seq) of Hmga2 wt and KO PSCs induced to differentiate into EpiLCs.

Hmga2基因敲除（KO）多能干细胞（pluripotent stem cells）无法分化为上胚层样干细胞（epiblast-like stem cells，EpiLCs）。本研究借助该实验体系，探究了EpiLCs诱导过程中的染色质动态变化，发现Hmga2的缺失会影响组蛋白修饰H3K27me3，该修饰在Hmga2 KO细胞中的水平更高。然而，分化过程中野生型（wild type，wt）与Hmga2 KO细胞的基因表达差异，未显示出多梳抑制复合体2（Polycomb Repressive Complex 2，PRC2）靶基因的显著富集。类似地，稳定培养的上胚层干细胞中内源性Hmga2与染色质的结合情况，也未与Hmga2 KO细胞中观察到的基因表达变化呈现明确关联。染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）结果证实，Hmga2蛋白优先结合与核纤层（nuclear lamina）相关的染色质区域。鉴于核纤层参与三维染色质结构的构建，我们进一步探究了Hmga2缺失对该过程的潜在影响。对野生型与Hmga2 KO细胞的Hi-C（高通量染色体构象捕获）数据进行分析后发现，Hmga2 KO细胞中的拓扑关联结构域（Topologically Associating Domain，TAD）间相互作用与野生型细胞存在差异，且这类差异明确表现为：与核纤层相关或无关的染色质区域的TAD间相互作用呈现独特的分区模式。本数据集包含三类组学数据：① 经诱导分化为EpiLCs的Hmga2野生型与KO多能干细胞的RNA测序（RNA-seq）比较基因表达谱分析数据；② 采用高通量测序技术开展EpiLCs中Hmga2的全基因组结合/占据谱分析的数据；③ 经诱导分化为EpiLCs的Hmga2野生型与KO多能干细胞的高通量染色体构象捕获测序（HiC-seq）数据。