The ARF tumor suppressor targets PPM1G/PP2Cγ to counteract NF-κB transcription tuning cell survival and the inflammatory response

Inducible transcriptional programs mediate the regulation of key biological processes and organismal functions. Despite their complexity, cells have evolved mechanisms to precisely control gene programs in response to environmental cues to regulate cell fate and maintain normal homeostasis. Upon stimulation with proinflammatory cytokines such as tumor necrosis factor-α (TNF), the master transcriptional regulator nuclear factor (NF)-κB utilizes the PPM1G/PP2Cγ phosphatase as a coactivator to normally induce inflammatory and cell survival programs. However, how PPM1G activity is precisely regulated to control NF-κB transcription magnitude and kinetics remains unknown. Here, we describe a mechanism by which the ARF tumor suppressor binds PPM1G to negatively regulate its coactivator function in the NF-κB circuit thereby promoting insult resolution. ARF becomes stabilized upon binding to PPM1G and forms a ternary protein complex with PPM1G and NF-κB at target gene promoters in a stimulidependent manner to provide tunable control of the NF-κB transcriptional program. Consistently, loss of ARF in colon epithelial cells leads to up-regulation of NF-κB antiapoptotic genes upon TNF stimulation and renders cells partially resistant to TNFinduced apoptosis in the presence of agents blocking the antiapoptotic program. Notably, patient tumor data analysis validates these findings by revealing that loss of ARF strongly correlates with sustained expression of inflammatory and cell survival programs. Collectively, we propose that PPM1G emerges as a therapeutic target in a variety of cancers arising from ARF epigenetic silencing, to loss of ARF function, as well as tumors bearing oncogenic NF-κB activation.Fil: Hyder, Usman. University of Texas; Estados UnidosFil: McCann, Jennifer L.. University of Texas; Estados UnidosFil: Wang, Jinli. University of Texas; Estados UnidosFil: Fung, Victor. University of Texas; Estados UnidosFil: Bayo Fina, Juan Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: D'Orso, Iván. University of Texas; Estados Unido

KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II

SummaryThe transition from transcription initiation to elongation at promoters of primary response genes (PRGs) in metazoan cells is controlled by inducible transcription factors, which utilize P-TEFb to phosphorylate RNA polymerase II (Pol II) in response to stimuli. Prior to stimulation, a fraction of P-TEFb is recruited to promoter-proximal regions in a catalytically inactive state bound to the 7SK small nuclear ribonucleoprotein (snRNP) complex. However, it remains unclear how and why the 7SK snRNP is assembled at these sites. Here we report that the transcriptional regulator KAP1 continuously tethers the 7SK snRNP to PRG promoters to facilitate P-TEFb recruitment and productive elongation in response to stimulation. Remarkably, besides PRGs, genome-wide studies revealed that KAP1 and 7SK snRNP co-occupy most promoter-proximal regions containing paused Pol II. Collectively, we provide evidence of an unprecedented mechanism controlling 7SK snRNP delivery to promoter-proximal regions to facilitate "on-site" P-TEFb activation and Pol II elongation

Host Cell Factors in HIV Replication: Meta-Analysis of Genome-Wide Studies

We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an “encyclopedia” of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets

RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation.

The transition from transcription initiation to elongation at the HIV-1 promoter is controlled by Tat, which recruits P-TEFb to TAR RNA to phosphorylate RNA polymerase II. It has long been unclear why the HIV-1 promoter is incompetent for elongation. We report that P-TEFb is recruited to the promoter in a catalytically inactive state bound to the inhibitory 7SK small nuclear ribonucleoprotein (snRNP), thereby preventing elongation. It also has long been believed that TAR functions to recruit Tat to the promoter, but we find that Tat is recruited to the DNA template before TAR is synthesized. We propose that TAR binds Tat and P-TEFb as it emerges on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase. Recruitment of an inhibitory snRNP complex at an early stage in the transcription cycle provides a new paradigm for controlling gene expression with a noncoding RNA

Genome-wide analysis of KAP1, the 7SK snRNP complex, and RNA polymerase II

The transition of RNA polymerase II (Pol II) from transcription initiation into productive elongation in eukaryotic cells is regulated by the P-TEFb kinase, which phosphorylates the C-terminal domain of paused Pol II at promoter-proximal regions. Our recent study found that P-TEFb (in an inhibited state bound to the 7SK snRNP complex) interacts with the KAP1/TRIM28 transcriptional regulator, and that KAP1 and the 7SK snRNP co-occupy most gene promoters containing paused Pol II. Here we provide a detailed experimental description and analysis of the ChIP-seq datasets that have been deposited into Gene Expression Omnibus (GEO): GS72622, so that independent groups can replicate and expand upon these findings. We propose these datasets would provide valuable information for researchers studying mechanisms of transcriptional regulation including Pol II pausing and pause release. Keywords: P-TEFb/7SK snRNP, KAP1, RNA polymerase II, ChIP-seq, Transcription elongatio

Targeting Tat Inhibitors in the Assembly of Human Immunodeficiency Virus Type 1 Transcription Complexes▿ †

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein, which relieves a block to elongation by recruiting an elongation factor, P-TEFb, to the viral promoter. Here, we report the discovery of potent Tat inhibitors that utilize a localization signal to target a dominant negative protein to its site of action. Fusing the Tat activation domain to some splicing factors, particularly to the Arg-Ser (RS) domain of U2AF65, creates Tat inhibitors that localize to subnuclear speckles, sites where pre-mRNA processing factors are stored for assembly into transcription complexes. A U2AF65 fusion named T-RS interacts with the nonphosphorylated C-terminal domain of RNA polymerase II (RNAP II) via its RS domain and is loaded into RNAP II holoenzyme complexes. T-RS is recruited efficiently to the HIV-1 promoter in a TAR-independent manner before RNAP II hyperphosphorylation but not to cellular promoters. The “preloading” of T-RS into HIV-1 preinitiation complexes prevents the entry of active Tat molecules, leaving the complexes in an elongation-incompetent state and effectively suppressing HIV-1 replication. The ability to deliver inhibitors to transcription complexes through the use of targeting/localization signals may provide new avenues for designing viral and transcription inhibitors

Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA.

Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription