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

Abl kinase deficiency promotes AKT pathway activation and prostate cancer progression and metastasis

AbstractAbl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation, motility, and in vivo tumor growth in some settings. To investigate the role of Abl kinases in prostate cancer, we generated Abl-deficient cells in a pre-clinical model of spontaneously metastatic, androgen-indifferent prostate cancer. Loss of Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro. Treatment of Abl kinase-expressing cells with the Abl kinase inhibitor imatinib phenocopied the malignant phenotypes observed in Abl-deficient tumor cells. In addition, inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells. Our data reveal that Abl family kinases can function as suppressors of prostate cancer progression and metastasis by restraining AKT signaling, a signaling pathway known to be associated with emergence of metastatic castration-resistant prostate cancer.</jats:p

Supplementary Information from α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

&lt;p&gt;RNAi targeting sequences, Supplemental Figure Legends, &amp; Supplemental Figures S1-S11&lt;/p&gt;</jats:p

Data from α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

&lt;div&gt;Abstract&lt;p&gt;Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an &lt;i&gt;in vivo&lt;/i&gt; model of prostate cancer metastasis via depletion of α3β1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3β1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3β1-Abl kinase-Hippo suppressor pathway identified α3 integrin–deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. &lt;i&gt;Cancer Res; 76(22); 6577–87. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;</jats:p