HMBA releases P-TEFb from HEXIM1 and 7SK snRNA via PI3K/Akt and activities HIV transcription

Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, we demonstrate that HMBA activates transiently the PI3K/Act pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactivecomplex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited tothe HIV promotoer to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK sn RNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorrylated and released from P-TEFb and 7SK snRNA via the PI3K/Act pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation

Inhibition of CDK12 elevates cancer cell dependence on P-TEFb by stimulation of RNA polymerase II pause release

P-TEFb and CDK12 facilitate transcriptional elongation by RNA polymerase II. Given the prominence of both kinases in cancer, gaining a better understanding of their interplay could inform the design of novel anti-cancer strategies. While down-regulation of DNA repair genes in CDK12-targeted cancer cells is being explored therapeutically, little is known about mechanisms and significance of transcriptional induction upon inhibition of CDK12. We show that selective targeting of CDK12 in colon cancer-derived cells activates P-TEFb via its release from the inhibitory 7SK snRNP. In turn, P-TEFb stimulates Pol II pause release at thousands of genes, most of which become newly dependent on P-TEFb. Amongst the induced genes are those stimulated by hallmark pathways in cancer, including p53 and NF-κB. Consequently, CDK12-inhibited cancer cells exhibit hypersensitivity to inhibitors of P-TEFb. While blocking P-TEFb triggers their apoptosis in a p53-dependent manner, it impedes cell proliferation irrespective of p53 by preventing induction of genes downstream of the DNA damage-induced NF-κB signaling. In summary, stimulation of Pol II pause release at the signal-responsive genes underlies the functional dependence of CDK12-inhibited cancer cells on P-TEFb. Our study establishes the mechanistic underpinning for combinatorial targeting of CDK12 with either P-TEFb or the induced oncogenic pathways in cancer.Peer reviewe

Sequencing of freshly produced RNA following exposure of cells to DNA damage-inducing UV mimetic 4-hydroxyaminoquinolone (4-NQO)

We used Illumina-HiSeq4000 to sequence 4sU-labelled RNA samples isolated from unchallenged and DNA damaged HeLa Flp-In cells, which revealed the nature of transcriptional response folowing genotoxic stress and the contribution of P-TEFb kinase in DNA damage-induced gene transcription.We mock treated or treated HeLa Flp-In cells for 1 or 2 hr with DMSO, 4-NQO, or 4-NQO + flavopiridol (FP) as indicated below. During the last 30 minutes of the treatments, we labeled the RNA or not with the nucleoside analogue 4-thiouridine (500µM 4sU) for 30 minutes.Bugai A, Quaresma AJC, Friedel CC, Lenasi T et al. P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress. Mol Cell 2019 Apr 18;74(2):254-267.e10. PMID: 3082437