Function Of PARP1 In Transcription Elongation

The eukaryotic transcription mediated by RNA polymerase ?(Pol ?)consists of several stages,including initiation,elongation and termination.Since the identification of the Positive-Transcription Elongation Factor b(P-TEFb),the regulation of transcription elongation has drawn more and more attention.Consisting of Cyclin Dependent Kinase 9(CDK9)and its binding partner Cyclin T1(CycT1),PTEFb can hyperphosphorylate the C-terminal domain(CTD)of the largest subunit of Pol ? to antagonize the inhibitory effect of negative elongation factors and thus release Pol ? from promoter proximal pausing,optimize its elongation activity.Due to its important role in transcription elongation,the function of P-TEFb has been tightly regulated by a series of positive and negative factors,which form a dynamic PTEFb network,including:(1)7SK snRNP where P-TEFb activity is inhibited;(2)Super Elongation Complex(SEC)where P-TEFb and ELL2 work together to stimulate transcription elongation;(3)Brd4-P-TEFb.It has been a long time since the identification of the components of P-TEFb network by our group and others.Although a lot of effort has been put into searching for new binding factors ever since,little progress is made.Other than identification of new associating factors,study of post-translational modification can also help better understand the regulation of PTEFb function.In this project,we discuss the identification and characterization of ADPribosylation on CycT1,we found that the ADP-ribosylation depends on the HRD on CycT1 and inhibits P-TEFb activity.First,we found that among the key transcription elongation factors CDK9,CycT1,ELL1 and ELL2,only CycT1 was found to be ADP-ribosylated.Next,the modification on CycT1 was identified as(poly)ADPribosylation(PARylation),which we discovered was medicated by ADPribosyltransferase PARP1.Meanwhile,in vitro PARylation reaction confirmed that CycT1 could be modified by PARP1.Next,we found that the 433-533 region of CycT1,especially the Histidine Rich Domain(HRD)within this region,is required for its PARylation.Moreover,replacing Histidine residues within HRD to either Alanine,Lysine or Arginine,inhibits the transcription activity of P-TEFb as well as PARylation on CycT1.Inspired by the discovery,we further found that PARP1 targets and modifies transcription-engaged P-TEFb which are likely the same ones that undergo phase separation.Furthermore,we found that auto-PARylated PARP1 binds to CycT1-IDR in a HRD-dependent manner and promotes its phase separation,resulting in the co-localization of PARP1 and CycT1 in the liquid droplets.In vitro kinase assay revealed that PARylation inhibits P-TEFb's ability to hyperphosphorylate CTD52.Cosistently,H2O2 treatment,which induces the PARylation on CycT1,inhibits HIV-1 transcription which is regulated directly by P-TEFb.