Study On The Dynamic Changes And Modifications In The Process Of P-TEFb Activation

The transcription process of eukaryotic genes mainly includes the following steps:pre-initiation complex assembling,initiation,pausing and release,elongation,and termination.Pausing and release is the rate-limiting step and one of the most important regulatory step in the transcription process of eukaryotic genes.Nowadays,this step is known to be controlled by Positive Transcription Elongation Factor b(P-TEFb).Intracellular P-TEFb exists in both active and inactive forms.Inactive P-TEFb is present in 7SK-snRNP and binds to its inhibitory protein HEXIM1 thus losing kinase activity.Active P-TEFb is mainly bound to transcription factors like Brd4,SEC for releasing the pause.To the researchers’ knowledge about the regulation of P-TEFb activity to date,there is a dynamic yin-yang balance between the inactive P-TEFb complex and the active one,but how the inactive P-TEFb is converted into active P-TEFb,in which the molecular mechanisms and modifications are still not known.In this study,HeLa,HCT116,F1C2 and other cell lines were used as experimental materials,and HMBA treatment was used as a method for activating P-TEFb to convert inactive P-TEFb into active P-TEFb.An improved nuclear fractionation protocol was used as the basic experimental method,combined with immunoprecipitation,real-time quantitative PCR,CHIP-seq and other experimental techniques,we carry out in-depth and detailed research on the dynamic process of the conversion of inactive P-TEFb into active P-TEFb.We found that in the process of activation of P-TEFb,accompanied by the disruption of the 7SK-snRNP complex,the two subunits of the core P-TEFb,Cdk9 and CyclinT,further dissociated.This dissociation is due to the dephosphorylation of Cdk9 T186 site by PP1α.We also found that the transfer of P-TEFb from chromatin free components LSF to chromatin bound components HSF is an important marker of P-TEFb activation.Inhibition of PPla can block this process and inhibit the formation of Brd4-P-TEFb and SEC-P-TEFb complex.It is indicated that activated P-TEFb does not form a recruitable P-TEFb complex and involved in the transcriptional cycle in an unlimited way.The way P-TEFb functions may be in turn forming active complex and inactive complex.In addition,after the dissociation of Cdk9 and CyclinT under stress,activation of P-TEFb require the interaction of Brd4,SEC with Cdk9 T-loop to help them restore binding capacity,thereby forming a complete P-TEFb molecule and recruitable P-TEFb complexes,thus restore transcriptional activity.Brd4,SEC is able to increase the autophosphorylation activity of Cdk9 in vitro.The mimicing polypeptide of Cdk9 T-loop can effectively block the interaction of Brd4,SEC with Cdk9,thereby inhibiting the activation process of P-TEFb and reducing the expression level of P-TEFb-dependent pausing gene.Our study unveiled the dynamic molecular changes and modifications during the activation of P-TEFb for the first time,and found two important targets PP1α and Cdk9 T-loop,which can block the activation process of P-TEFb.We provide a theoretical basis for the specific inhibitor of P-TEFb and the treatment of P-TEFb activity-related diseases.