Two-pronged Binding with Bromodomain-containing Protein 4 Liberates Positive Transcription Elongation Factor b from Inactive Ribonucleoprotein Complexes

Abstract

The positive transcription elongation factor b (P-TEFb) exists in two forms in cells as follows: an inactive form where the core components cyclin T1 and CDK9 are incorporated in the 7SK small nuclear ribonucleoprotein complex containing the inhibitory molecule HEXIM1, and an active form, part of which associates with the bromodomain-containing protein BRD4. Here, we define a novel interaction between P-TEFb and BRD4 involving tri-acetylated cyclin T1 (acK380, acK386, and acK309) and the second bromodomain in BRD4. This interaction is observed with the short splice variant of BRD4 (amino acids 1-722) lacking a previously defined C-terminal P-TEFb-interacting domain (PID). Notably, P-TEFb complexes associated with short BRD4 contain HEXIM1 and 7SK snRNA, implicating the PID in the liberation of P-TEFb from the 7SK small nuclear ribonucleoprotein complex (7SK snPNP). Overexpression of the PID alone in cells dissociates HEXIM1 and 7SK snRNA from P-TEFb, but it is not sufficient to activate P-TEFb-dependent transcription of the HIV LTR. Our data support a model where two BRD4 domains, the second bromodomain and the PID, bind P-TEFb and are required for full transcriptional activation of P-TEFb response genes.