新型组蛋白去甲基化酶及甲基化调控方式的筛选和鉴定

Histone methylation can be removed by demethylases. Up to now, two classes of about 20 histone demethylases specific for different lysine residues were identified, but demethylases for several other lysine residues have not been found. Also, a demethylase activity against the arginine residue has not been convincingly demonstrated. To identify new histone demethylases, we performed high content cell-based screening. In this screening, we transfected each cDNA encoding 2500 nuclear proteins with more than 300 amine-acids in length into cells and obtained hits that could reduce the levels of several specific methylations in lysine and arginine residues in transfected cells by immunocytochemistry. Through western blot analysis on transfected and non-transfected cells as the secondary screening, we narrowed down the number of hits as the candidates. The candidates were expressed and purified from bacteria or insect cells, and reacted with bulk histones with various methylations or histone peptides with specific methylations in the presence or absence of various set of cofactors. The reaction was subjected to western blot analysis or mass spectrometry analysis to examine if the candidates could remove the specific methylation from bulk histones or histone peptides. We are now in the process of confirming the demethylase activity by demethylation product characterization, domain mapping, and crossing out of cofactors.

组蛋白甲基化是一种重要的表观遗传修饰方式，与个体发育和疾病密切相关。近几年来，组蛋白赖氨酸去甲基化酶的不断发现证明了赖氨酸甲基化是一种可逆的过程，但H4K20的组蛋白赖氨酸去甲基化酶仍未找到，精氨酸去甲基化酶也未找到。我们在筛选和鉴定组蛋白赖氨酸去甲基化酶的基础上，提出用半高通量方法筛选H4K20的组蛋白赖氨酸去甲基化酶和组蛋白精氨酸去甲基化酶：用转染的方法逐个把2500个表达细胞核蛋白质的质粒转入真核细胞中使其高表达，然后用免疫荧光染色的方法寻找能使目标甲基化水平降低的蛋白质，从而获得候选去甲基化酶；表达纯化候选酶，进行体外酶活实验，确定在没有其它蛋白质的帮助下，候选酶本身就有去除组蛋白甲基化的能力；通过对辅助因子和突变体的研究，鉴定其催化机制；还将用条件性敲除小鼠模型研究其与个体发育和癌症的关系，以揭示其生物学功能。该研究结果将对表观遗传领域和其它生物医学研究领域产生影响。

组蛋白甲基化是一种重要的表观遗传修饰方式，与个体发育和疾病密切相关。近几年来，组蛋白赖氨酸去甲基化酶的不断发现证明了赖氨酸甲基化是一种可逆的过程，但H4K20的组蛋白赖氨酸去甲基化酶仍未找到，精氨酸去甲基化酶也未找到。我们在筛选和鉴定组蛋白赖氨酸去甲基化酶的基础上，用高通量方法筛选并鉴定了H4K20的组蛋白赖氨酸去甲基化酶，免疫印迹实验和质谱分析发现纯化的2A4在体外可以去除H4K20me1/2/3，并产生甲醛；ChIP-Seq实验发现2A4在细胞内可以去除H4K20me1/2/3；还发现2A4通过去除H4K20甲基化激活重复序列的转录；敲低2A4导致H4K20甲基化的去除延迟，细胞周期延长，细胞分裂变慢。由于2A4蛋白质序列与LSD和JmjC家族没有保守性，它是一类新型的组蛋白赖氨酸去甲基化酶。.同样方法我们获得一个组蛋白精氨酸去甲基化酶候选蛋白质1G11。1G11在细胞内高表达可以降低精氨酸甲基化水平。免疫印迹实验显示纯化的1G11蛋白质在体外可以降低精氨酸甲基化水平，并呈现浓度依赖效应。目前，我们正在鉴定1G11酶反应的特异性、去甲基化产物、催化结构域和催化机制，并用结构生物学方法进一步揭示其催化机理。如果证实，1G11将是第一个组蛋白精氨酸去甲基化酶。