GATA因子转换失调在苯代谢物抑制红系分化过程中的作用

The erythropoietic depression has been widely observed in benzene-exposed human and animal individuals, but little is known about its mechanism. Our precious study has shown that phenolic metabolites of benzene inhibited erythroid differentiation of K562 cells, accompanied by decreased expression of erythroid specific transcription factors GATA2 and GATA1, and reduced GATA1 occupany of GATA motifs in globin gene loci. During erythroid differentiation, GATA1 and GATA2 show dynamic changes in their expression profiles, and the dominant GATA factor switched from GATA2 to GATA1, which is referred to as the “GATA Factor Switching”. The GATA Factor Switching leads to changes in the occupancy of GATA motifs in regulatory regions of erythroid genes from GATA2 to GATA1, and GATA motifs as a switch (GATA-Switch) control their expression. Undoubtedly, disturbed GATA Factor Switching will lead to abnormal erythroid differentiation. In present project, it is to be addressed whether GATA Factor Switching occurs, and GATA motifs in erythroid genes function as switchs to control erythroid differentiation in benzene metabolites-exposed erythroid progenitors when the progenitors are treated with erythroid differentiation inducer, which will provide evidence to elucidate the underlying mechanism of erythroid toxicity of benzene.

红系造血抑制是苯血液毒性主要表现，其机制还不明确。申请者前期研究发现，苯的酚类代谢物短期处理能显著抑制K562细胞的红系分化，并抑制红系分化关键转录因子GATA2和GATA1的表达，降低GATA1在珠蛋白基因的占领。而在红系分化过程中，GATA2和GATA1的表达会发生所谓“GATA因子转换”，造成红系分化关键基因的GATA位点结合的GATA2被GATA1替换，GATA位点发挥开关作用巧妙编排这些基因的表达变化，保证红系分化按照一定的程序有序进行。因此，GATA因子转换失调势必影响GATA开关的有效开启，进而干扰红系分化。项目拟观察苯代谢物处理的红系祖细胞在诱导红系分化时GATA因子转换是否准确实现，红系分化关键基因的GATA位点能否有效发挥开关作用以调节基因的有序表达或沉默，从而探讨GATA因子转换和GATA开关在苯代谢物抑制红系分化中的作用，以充分认识苯的红系血液毒性机制。

红系造血抑制是苯血液毒性主要表现，其机制还不明确。GATA2和GATA1的表达发生所谓从GATA2到GATA1转换在红系分化过程发挥关键作用。项目研究了苯代谢物氢醌处理的K562细胞在诱导红系分化时GATA因子转换情况，从而探讨GATA因子转换在苯代谢物抑制红系分化中的作用。结果发现，从苯代谢物氢醌长期处理3周的K562细胞中克隆筛选出细胞株HQ1的红系分化能力低下；该细胞株在Hemin诱导不同时间（1-5天）的情况下GATA1因子的表达显著低于对于相应对照细胞，并与红系分化相关的NF-E2、珠蛋白基因、血红素合成酶相关基因和红系分化细胞表面标志基因的表达规律一致，而GATA2因子出现持续高表达，这些变化与GATA1和GATA2因子在这些基因上的募集水平的变化规律相一致，而DNA甲基化抑制剂（5-Aza-CdR）和组蛋白去乙酰化抑制剂（TSA）可有效将阻止这些变化；氢醌处理3天的K562细胞也出现类似与HQ1细胞株的变化。进一步利用ChIP-seq全基因组范围分析GATA1和2占位情况显示，Hemin诱导可显著提高GATA1因子占领的基因数量，而减少GATA2因子占领的基因数量，氢醌处理可显著抑制Hemin诱导的GATA1和2因子占领基因数量变化；利用RNA-Seq和液相色谱-质谱联用技术分析全基因转录组和蛋白质组学变化，Hemin可诱导的相当一部分基因表达的上调或下调，而氢醌处理对Hemin的作用具有抑制作用；进一步从红系分化相关基因角度进行深度组学分析，氢醌可显著抑制Hemin诱导的GATA1或GATA2因子在这些基因的占领改变，并与基因的表达变化相一致。结果表明，氢醌通过改变红系分化过程中GATA因子占位水平，上调GATA2转录和蛋白水平，使Hemin诱导的GATA因子转换失调，从而下调了红系分化相关基因转录和蛋白表达，从而抑制血红蛋白合成和红系分化进程。