Mechanisms And Founctions Of P-TEFb Complex In Autosomal Dominant Polycystic Kidney Disease

Objective: 1.To explore the molecular mechanisms underlying the activation of positive transcription elongation factor b(P-TEFb)and its roles in autosomal dominant polycystic kidney disease(ADPKD).2.To explore the therapeutic effect of targeting P-TEFb in ADPKD.Methods and results: This project was divided into four parts: Part one: To identify novel therapeutic targets of ADPKD,we performed a chemical library screening,which included transcriptional and epigenetic inhibitors,in an embryonic kidney cyst model.The screening identified flavopiridol(FP)as a potent inhibitor of cyst formation in mouse embryonic kidney.Furthermore,P-TEFb inhibition(FP treatment or CDK9 knockdown)also showed potent inhibitory effect in another cystogenesis assay,an established 3D-MDCK cyst model.Part two: To examine the activity of P-TEFb complex in polycystic kidneys in vivo and explore if P-TEFb can be a targeting candidate for ADPKD treatment.We first performed western blotting/ immunofluorescence and RT-q PCR assays to determinate the amount of free P-TEFb(active P-TEFb)in ADPKD kidney tissues.The results demonstrated that the activity of P-TEFb significantly increased in the kidneys from medium-term and long-term ADPKD mouse models and human patients.Next,we found that FP treatment slowed cyst growth by inhibiting cyst-lining epithelial cell proliferation.Furthermore,FP treatment improved kidney function by decreasing kidney injury,inflammation,and fibrosis in vivo.Part three: To understand the molecular mechanisms underlying P-TEFb activation in ADPKD,we performed a serious of biochemical experiments and zebrafish assays.The results demonstrated that hyperactived c AMP-PKA signaling disrupted the inactive P-TEFb/HEXIM1/7SK sn RNP complex by PKA-mediated phosphorylation of HEXIM1 at serine-158 and accelerated the disease progression.Part four: To investigate how hyperactived P-TEFb promoted cystogenesis in ADPKD,we performed Ch IP sequencing(Ch IP-seq)and RNA sequencing(RNA-seq)in mouse kidneys.Bioinformatics analysis showed that Pol II pausing index was decreased on the promoter-proximal region of cystogenesis-related genes in ADPKD,and it can be rescued by FP treatment.This indicates that the hyperactived P-TEFb increases the transcription elongation of cystogenesis-related genes,thereby accelerating disease progression.Conclusion: In this study,we established a critical role for P-TEFb in controlling cystogenic gene transcription.A chemical screenning identified flavopiridol,an inhibitor of P-TEFb,as a potent anti-cystogenesis agent.P-TEFb was hyperactivated in mouse and human ADPKD kidneys.Genetic activation of P-TEFb promoted pronephric cyst formation in a zebrafish ADPKD model.Pharmacological inhibition of P-TEFb attenuated cyst development in the medium-term and long-term ADPKD mouse models.Mechanistically,activation of c AMP-PKA signaling led to phosphorylation of HEXIM1 at serine 158.The hyperphosphorylation of HEXIM1 in ADPKD disrupted inactive P-TEFb/HEXIM1/7SK sn RNP complex and released active P-TEFb.Further genome-wide analysis demonstrated that P-TEFb inhibition suppressed a pathologic gene expression program by abrogating abnormal transcriptional pause release.This study implicates aberrant activation of P-TEFb-mediated transcription elongation during cystogenesis and identifies P-TEFb as a potential therapeutic target in ADPKD.