Functional Association of Gdown1 with RNA Polymerase II Poised on Human Genes

Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.National Human Genome Research Institute (U.S.) (Grant HG002668-05

The Mechanism of Release of P-TEFb and HEXIM1 from the 7SK snRNP by Viral and Cellular Activators Includes a Conformational Change in 7SK

The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb

The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.

<p>HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#s4" target="_blank">Materials and Methods</a>. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.</p

Model of P-TEFb and HEXIM1 release from the 7SK snRNP.

<p>P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.</p

Release of P-TEFb from the 7SK snRNP by the P-TEFb binding domain of Brd4.

<p>A) A schematic of Brd4 constructs used. BD1, Bromodomain 1; BD2, Bromodomain 2; ET, Extraterminal domain; H1, H2 and H3, Helical domains 1, 2 and 3. Brd4 1209–1362 contains three helical regions, Brd4 1209–1362 Δ1329–1345 is missing helical region 3 which is required for P-TEFb binding. B) Recombinant protein expressed and purified from <i>E. coli</i>. A contaminating band can be seen in the Brd4 mutant preparation and this was confirmed to be an <i>E. coli</i> protein by Western blot (data not shown), not full length Brd4. M, Marker; 1, Brd4 1209–1362; 2, Brd4 1209–1362 Δ1329–1345. C) Indicated amounts of Brd4 were added into the release reaction for the indicated times. D) Same as in C except the Brd4 mutant missing helical domain 3 was used for the reactions. E) Brd4 release was quantified from three independent experiments. Two independent experiments were done to calculate the mean for the Brd4 helical domain 3 mutant. The y-axis is a measure of percent of Cdk9 left in the complex. All error bars represent standard error.</p