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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.

SUBMITTER: Schroder S

PROVIDER: S-EPMC3256921 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-?B mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

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Two-pronged binding with bromodomain-containing protein 4 liberates positive transcription elongation factor b from inactive ribonucleoprotein complexes.

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