应用TALEN技术研究LSD1在造血分化中对GATA switch的调控机制

LSD1, the first well-characterized H3K4 demethylase, plays an important role in hematopoiesis. Based on our preliminary data, we hypothesize that LSD1 mediated epigenetic modification regulates the switching of GATA factors during hematopoietic differentiation. It has been reported that GATA2 is expressed in hematopoietic stem cells (HSCs) and its expression becomes gradually decreased upon erythroid differentiation, while GATA1 is absent in HSC stage and is upregulated upon differentiation. The interplay between GATA1 and GATA2 is critical for maintaining homeostasis of cells during HSC differentiation. We propose to employ ChIP-seq, GST Pull-down and co-immunoprecipitation (co-IP) to investigate if GATA2 recruits LSD1 to repress GATA1 in HSCs and whether the inhibition of GATA1 expression upon erythroid differentiation is dependent on LSD1 and its mediated histone demethylation. In addition, to understand the functional effects of LSD1 during HSC differentiation, we will use TALEN-mediated knockout of LSD1 to test our hypothesis that LSD1 ablation impairs HSCs homeostasis and erythroid differentiation by disrupting the normal expression pattern of GATA1 and GATA2. The proposed experiments will provide a new insight into epigenetic mechanism by which LSD1 regulates the GATA switch during hematopoiesis and establish a TALEN mediated gene KO model in hematopoietic cells.

LSD1是第一个组蛋白去甲基化酶，本研究拟应用TALEN靶向基因操作技术对基因组进行定点改造，构建LSD1缺失细胞，探究LSD1在造血分化中对GATA Switch的调控机制。GATA2主要在造血干/祖细胞的增殖和分化中发挥作用，并随造血细胞分化表达下调，而GATA1表达上调，进而调控造血细胞的增殖与分化（GATA Switch）。项目拟通过ChIP-seq、GST-pull down和IP等技术揭示在造血早期GATA2募集LSD1至GATA1位点，抑制GATA1表达，但随造血分化TAL1与GATA1结合，招募LSD1至GATA2 位点，进而抑制GATA2表达；并在TALEN技术构建的LSD1缺失细胞中通过RT-qPCR及ChIP分析GATA转录因子表达及组蛋白甲基化水平的变化，明确LSD1对GATA Switch的调控机制，从而建立应用TALEN技术研究LSD1调控造血分化机制的新模型。

GATA2转录因子主要表达于造血干细胞和祖细胞（HS/pc），并协同特定转录因子GATA1调节造血细胞的增殖与分化。然而GATA转录因子如何协同组蛋白去甲基化酶LSD1调控造血细胞分化的机制尚不明确。本研究表明，沉默的LSD1会增加GATA2表达并抑制小鼠红系白血病细胞MEL和胚胎干细胞分化。此外，造血分化早期GATA2募集LSD1至GATA1基因的dbG、IE位点，通过LSD1去甲基化功能抑制GATA1表达；随造血细胞分化，GATA2和LSD1相互结合减弱，TAL1和LSD1相互结合增强，TAL1将LSD1招募至GATA2基因的1S启动子位点，进而抑制GATA2表达，促进红细胞分化。LSD1通过调节GATA1、GATA2蛋白表达对造血系统分化发挥重要调控功能，LSD1、TAL1、GATA2以复合体形式动态调控这一过程。本研究从一个全新的组蛋白修饰的角度阐释了GATA Switch的调控机制，明确了LSD1所介导的表观遗传学修饰对GATA Switch的调控功能，从而建立了LSD1调控造血分化机制的新模型。