AFF4在人类HEL细胞中广泛地影响RNA聚合酶Ⅱ的停滞释放  

AFF4 globally affects the release of paused RNA polymerase Ⅱ in HEL cells

作　　者：杨子玫 张格 魏刚 经莉莉 于明 Zimei Yang;Ge Zhang;Gang Wei;Lili Jing;Ming Yu(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China;CAS Key Laboratory of Computational Biology,Shanghai Institute of Nutrition and Health,Chinese Academy of Sciences,University of Chinese Academy of Sciences,Shanghai 200031,China;School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;Department of Pathology,Shanghai Chest Hospital,Shanghai Jiao Tong University,Shanghai 200052,China)

机构地区：[1]上海交通大学药学院,上海200240 [2]中国科学院上海营养与健康研究所,中国科学院计算生物学重点实验室,中国科学院大学,上海200031 [3]上海交通大学生命科学技术学院,上海200240 [4]上海交通大学附属上海胸科医院病理科,上海200052

出　　处：《遗传》2023年第8期658-668,共11页Hereditas（Beijing)

摘　　要：在转录延伸过程中,P-TEFb激酶(由激酶CDK9和Cyclin T1组成的二聚体)是释放停滞的Pol Ⅱ这一步的关键调节因子。在人类细胞中,它可参与形成三个更大的复合体,即超级延伸复合体(super elongation complex,SEC)、BRD4/P-TEFb和7SKsnRNP,其活性在前两者中不被抑制,而在后者中被抑制。除P-TEFb外,SEC还含有AFF1或4、AF9或ENL、ELL1、2或3,EAF1或2等无酶活亚基,这些亚基被认为可通过P-TEFb影响转录延伸。本课题组前期的研究和其他实验室的研究工作均已表明AFF1、AF9和ENL亚基均可调控转录启始,但尚不清楚AFF4是否具有相似的功能。在调控基因表达的选择性方面,近期有研究表明BRD4/P-TEFb在人类结直肠腺癌细胞DLD-1中主要负责除热激基因以外的其他基因的停滞释放,而SEC负责热激基因的停滞释放。为了深入理解AFF4的功能,本研究利用RNA干扰技术在人类红系白血病细胞HEL中敲低了AFF4,发现RNA聚合酶Ⅱ (Pol Ⅱ)的羧基端重复区(C-terminal domain,CTD)内第二个丝氨酸(Serine 2,Ser-2)的磷酸化水平降低。利用RNA-seq和CUT&Tag确定了AFF4的直接靶基因,利用ChIP-seq和PRO-seq发现AFF4对转录启始的影响不明确、对Pol Ⅱ的停滞释放的调控不局限于热激基因,利用ChIP-qPCR发现AFF4缺失降低了P-TEFb的染色质结合。上述研究结果表明,AFF4对Pol Ⅱ的停滞释放的调控可能具有一定的细胞类型特异性,即在一些细胞内仅调控热激基因的停滞释放,而在另一些细胞中调控的对象却不仅限于热激基因。P-TEFb,a heterodimer of the kinase CDK9 and Cyclin T1,is a critical regulator of promoter-proximal pause release of Pol Ⅱ in metazoans.It is capable of forming three larger complexes,including the super elongation complex(SEC),the BRD4/P-TEFb complex and the 7SK snRNP.In the SEC or the BRD4/P-TEFb complex,P-TEFb is enzymatically active,while in the 7SK snRNP,its activity is inhibited.The SEC consists of AFF1 or 4,ENL or AF9,ELL1,2 or 3 and EAF1 or 2 in addition to P-TEFb,the only subunit with catalytic activity,and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb.We and others recently found that AFF1,ENL and AF9 are capable of regulating transcriptional initiation,but it is unknown yet whether AFF4 is also capable of doing so.With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex,one recent study showed that in human DLD-1 cells,the SEC only regulates pause release of heat shock(HS)genes,whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes.However,it is unclear whether those mechanisms are general.In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation,we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol Ⅱ.Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag.Notably,we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol Ⅱ on several hundred HS and thousands of non-HS genes.Mechanistically,AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol Ⅱ.These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

关 键 词：P-TEFB 混合系白血病 超级延伸复合体 AFF4 转录延伸 

分 类 号：R394[医药卫生—医学遗传学]