核纤层蛋白调控染色质结构促胚胎干细胞分化的机制研究

干细胞分化的本质是基因的选择性表达，基因表达依赖于染色质结构，但干细胞分化过程中染色质结构的动态变化规律及其调控机制仍不清楚。最近研究显示视磺酸能通过调控核纤层蛋白Lamin A的表达来诱导胚胎干细胞（ESC）分化。预实验基于组蛋白H3K9me3荧光能量共振转移（FRET）探针，发现视磺酸诱导ESC分化时异染色质标志H3K9me3整体动态下降，且早于干性基因的表达抑制。故提出科学假设：磷酸化的Lamin A促进H3K9me3去甲基化，介导染色质结构改变，促进ESC分化。本研究基于视磺酸诱导ESC分化的模型，以FRET探针为主要手段，结合CRISPR/Cas技术、Fluocell图像分析软件和Hi-C技术对Lamin A磷酸化、H3K9me3和染色质结构实时三维活细胞动态成像与调控，阐明ESC分化的染色质结构动态变化规律及其对ESC基因表达的调控。研究结果将为基因表达调控提供新理论和新思路。

Selective gene expression is crucial in stem cell differentiation. Gene expression is tightly regulated by chromatin structure. However, it is still unclear about the dynamics of chromatin structure and how it is regulated during stem cell differentiation. Recent study shows that retinoic acid (RA) induces embryonic stem cell (ESC) differentiation through the regulation of LMNA expression. Our preliminary results showed that H3K9me3, which was measured by histone H3K9me3 Fluorescence Resonance Energy Transfer (FRET) biosensor, decreased significantly during ESCs differentiation upon RA induction, and changed earlier than the reduction of pluripotent genes. In this proposal, we hypothesized that, Lamin A phosphorylation mediates demethylation of H3K9me3 in heterochromatin, which will dissociate heterochromatin from nuclear lamina and eventually change gene expression. The experimental model we will use is RA induced ESCs differentiation. We will utilize FRET biosensors as main technology, integrate with CRISPR/Cas gene editing system, Fluocell imaging analysis software, and Hi-C technology, to real-timely monitor and manipulate Lamin A phosphorylation and H3K9me3 dynamics as well as chromatin structure, which will elucidate chromatin dynamics and gene expression during ESC differentiation. We believe our results will provide new insights on the fundamental understanding of gene regulation.