A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation
- Yupeng Chen1,4,
- Lirong Zhang1,4,
- Conchi Estarás1,
- Seung H. Choi1,
- Luis Moreno Jr.1,
- Jonathan Karn2,
- James J. Moresco3,
- John R. Yates III3 and
- Katherine A. Jones1
- 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
- Corresponding author: jones{at}salk.edu
-
↵4 These authors contributed equally to this work.
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
Footnotes
-
Supplemental material is available for this article.
-
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.250449.114.
- Received August 5, 2014.
- Accepted September 16, 2014.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.