Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock
Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock
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DOI：
10.1128/mcb.23.21.7628-7637.2003
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发表时间：
2003-11-01
影响因子：
5.3
通讯作者：
Lis, JT
中科院分区：
文献类型：
--
作者：
Boehm, AK;Saunders, A;Lis, JT
未被诱导的果蝇HSP70基因将被快速激活。在此，我们用染色质免疫沉淀物的实时定量聚合酶链式反应和多线染色体免疫荧光技术，在体内检测了热休克时热休克因子(HSF)、RNA聚合酶II(POL II)及其磷酸化形式以及POL II激酶P-TEFb在体内的水平和位置的快速变化。这些研究捕捉了Pol II沿HSP70的募集和进展，并揭示了丝氨酸2和丝氨酸5磷酸化的不同时空模式：在未诱导的细胞中，启动子暂停的Pol II显示Ser5而不是Ser2磷酸化，在诱导细胞中，Ser2-P Pol II在启动子处的相对水平低于下游区域。热休克激活的早期时间点捕获了在P-TEFb之前招募到启动子的未磷酸化的POL11，在第一波转录中，可以看到POL11和P-TEFb激酶一起跟踪HSP70，动力学难以区分。POL 11在POL II暂停的其他几个基因上的分布显示出类似于HSP70的Ser5和Ser2磷酸化模式。这些对因子编排的研究在转录调控机制的建模方面设置了重要的限制。
The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol 11 recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol 11 and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol 11 distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.