ISL1在胚胎干细胞向心肌细胞分化过程中的表观遗传调控机制研究

Heart development is a complex process that relies on networks of interacting transcription factors. ISL1 is a member of LIM-homeodomain transcription factor families. It plays a pivotal role for the earliest stage of heart development. ISL1 mainly marks proliferating, undifferentiated progenitors of the second heart field (SHF). However, the mechanisms governing the gene expression of Isl1 and how ISL1 regulates the gene expression of its downstream targets during heart development are poorly defined. In this project, we are to exploit these scientific questions, focusing on the epigenetic regulatory mechanism of ISL1 in the control of gene expression during myocardium development. Our previous work (supported by NSFC grants) has demonstrated that the expression of Isl1 is activated by Wnt/beta-catenin pathway at the early stage and inhibited by Notch-Hes1 pathway at the middle-late stage of cardiac differentiation of ES cells. ISL1 can also recruit Brg1, Baf155 and Baf60 (components of SWI/SNF) to form a complex. ISL1 can interact with JMJD3 and SET-9. According to these clues and others reports, we propose a hypothesis that at the early stage of cardiac differentiation of ES cells, with the Wnt/beta-catenin pathway activated, p300/CBP is recruited by nuclear beta-catenin to the isl1 enhancer (-2300bp upstream ATG) to increase Isl1 transcription; at the middle-late stage, with the Notch pathway activated, PRC2, trimethylasing histone lysine 27, is recruited by Hes1 to the Isl1 promoter (-466bp to -481bp upstream ATG) to decrease Isl1 transcription. ISL1 regulates its downstream target genes (Nkx2.5, Mef2c, etc.) by recruiting beta-catenin and ATP-dependent chromatin-remodeling complex SWI/SNF as well the histone lysine 27 demethylase (JMJD3) to remove repressive H2K27me3 modifications, and the histone lysine 4 methyltransferase (SET-9) to add a modification (H3K4me) often associated with gene activation. Mouse ES cells (R1 cell line) will be used as a cardiac differentiation model. An inducible Tet-on lentivirus gene overexpression/RNAi system will also be employed to treat ES cells. The recruitment of P300/CBP, Hes1/PRC2 or SWI /SNF and SET-9/JMJD3 will be examined by ChIP-reChIP, Co-IP, GST pull-down and mass spectrometry, etc. The interaction domains will be defined by creating mutations or deletions in predicted domains. The involvement of WNT or Notch signaling pathway will be determined by standard methods. Methyltransferase activity will be examined. The results will help us to understand the epigenetic regulatory relationships on ISL1 during the cardiac differentiation process. It will also provide the information about cardiomyocyte reprogram and promote clinical research work on the regeneration medicine using stem cells.

ISL1是心脏发育关键分子之一，在发育过程呈动态表达。然而ISL1的动态表达调控机制及其对下游靶基因的具体调控方式尚未完全阐明。我们在前期工作基础上，提出如下假说：在胚胎干细胞向心肌分化过程中，Isl1基因动态表达受到WNT和Notch信号通路相互作用及其所介导的表观遗传机制调控；ISL1通过募集beta-catenin和染色质重塑复合物SWI /SNF，易化下游靶基因（Nkx2.5等）的染色质状态，并招募甲基化转移酶SET-9与去甲基化酶JMJD3，协同调节ISL1下游靶基因组蛋白H3K4的甲基化与H3K27的去甲基化状态，共同促进心肌特异性基因表达、促进向心肌细胞分化。我们将以ES细胞为研究体系，利用可诱导的Tet-on基因过表达/抑制系统，Co-IP和质谱等技术进一步验证该假说。本课题的完成，可为揭示ISL1在心肌细胞分化中的作用及机制和干细胞在心血管再生医学中的临床应用提供依据。

胰岛因子１（ISL1）是心脏发育关键分子之一，在发育过程呈动态表达。然而ISL1的动态表达调控机制及其对下游靶基因的具体调控方式尚未完全阐明。在本课题中，我们利用可诱导的Tet-on基因过表达/抑制系统，ChIP-Seq和RNA-Seq、Co-IP和质谱等技术在小鼠胚胎干细胞（ES细胞）向心肌细胞诱导分化的体系中，证明了在胚胎干细胞向心肌分化过程中，Isl1基因动态表达受到WNT和Notch信号通路相互作用及其所介导的表观遗传机制调控：在分化早期WNT信号中的b-catenin/LEF1/CBP复合体结合在Isl1基因增强子-2300bp和-2900bp处，促进其 H3K9的乙酰化水平提高ISL1的表达。在分化中晚期，Notch信号通路通过HES1-PRC2（EZH2）复合体结合在Isl1上游-466—-481bp位点，增加其组蛋白H3K27甲基化水平抑制ISL1的表达。同时，我们还阐明了在ES细胞向心肌细胞分化过程中，ISL1通过改变染色质状态和组蛋白甲基化修饰状态对下游靶基因进行调节的机制：ISL1通过募集b-catenin和染色质重塑复合物SWI /SNF，易化下游靶基因（Mef2c、Troponin-T等）的染色质状态，同时招募甲基化转移酶PHF8与去甲基化酶JMJD3，协同调节ISL1下游靶基因组蛋白H3K4的甲基化与H3K27的去甲基化状态，共同促进心肌特异性基因表达、促进向心肌细胞分化。本课题的完成，可为揭示ISL1在心肌细胞分化中的作用及机制和干细胞在心血管再生医学中的临床应用提供依据。