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Genes Dev. 2014 Oct 15;28(20):2261-75. doi: 10.1101/gad.250449.114.

A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation.

Author information

  • 1Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA;
  • 2Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA;
  • 3Department of Chemical Physiology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
  • 4Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA; jones@salk.edu.

Abstract

HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P-RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P-S2P transition at the integrated HIV-1 promoter.

KEYWORDS:

CTD phosphatase; HIV-1 Tat; Ssu72; elongation

PMID:
25319827
PMCID:
PMC4201287
DOI:
10.1101/gad.250449.114
[PubMed - indexed for MEDLINE]
Free PMC Article
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