G-actin Participates in RNA Polymerase II-dependent Transcription Elongation by Recruiting Positive Transcription Elongation Factor b (P-TEFb)

Ling Wang,Lei Zhai,Lijing Guo,Wen Tang,Xianlu Zeng,Tianyang Qi

doi:10.1074/jbc.m110.184374

Ling Wang, Lei Zhai + Show 4 more

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https://doi.org/10.1074/jbc.m110.184374

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Abstract

Actin is a key regulator of RNA polymerase (Pol) II-dependent transcription. Positive transcription elongation factor b (P-TEFb), a Cdk9/cyclin T1 heterodimer, has been reported to play a critical role in transcription elongation. However, the relationship between actin and P-TEFb is still not clear. In this study, actin was found to interact with Cdk9, a catalytic subunit of P-TEFb, in elongation complexes. Using immunofluorescence and immunoprecipitation assays, Cdk9 was found to bind to G-actin through the conserved Thr-186 in the T-loop. Overexpression and in vitro kinase assays showed that G-actin promotes P-TEFb-dependent phosphorylation of the Pol II C-terminal domain. An in vitro transcription experiment revealed that the interaction between G-actin and Cdk9 stimulated Pol II transcription elongation. ChIP and immobilized template assays indicated that actin recruited Cdk9 to a transcriptional template in vivo and in vitro. Using cytokine IL-6-inducible p21 gene expression system, we revealed that actin recruited Cdk9 to endogenous gene. Moreover, overexpression of actin and Cdk9 increased histone H3 acetylation and acetylized histone H3 binding to a transcriptional template through the interaction with histone acetyltransferase, p300. Taken together, our results suggested that actin participates in transcription elongation by recruiting Cdk9 for phosphorylation of the Pol II C-terminal domain, and the actin-Cdk9 interaction promotes chromatin remodeling.

Highlights

Of Ser-2 by Cdk9 promotes polymerase II (Pol II) to enter the productive elongation stage [3,4,5,6]
Actin Binds positive transcription elongation factor b (P-TEFb) in Elongation Complexes—Recent reports have shown that actin, acting as a component of hnRNP complexes, is coupled to Ser-2-phosphorylated Pol II C-terminal repeat domain (CTD) in active genes [33]
Immunoprecipitation assay with nuclear extract (NE) from untransfected or transfected HeLa cells (Fig. 1, D and E, respectively) was performed to confirm whether actin, Cdk9, and Pol II were present in the same complex

Summary

Introduction

Of Ser-2 by Cdk9 promotes Pol II to enter the productive elongation stage [3,4,5,6]. In addition, CTD phosphorylation is believed to help recruit factors involved in both elongation and chromatin remodeling [7]. Our data have provided convincing evidence that G-actin stimulated Pol II transcription elongation occurs via the recruitment of Cdk9 for phosphorylation of the Pol II CTD and that chromatin remodeling is promoted by the actin-Cdk9 interaction. Immunoprecipitation assay with NEs from untransfected or transfected HeLa cells (Fig. 1, D and E, respectively) was performed to confirm whether actin, Cdk9, and Pol II were present in the same complex.

Results

Conclusion