ABSTRACT: NELF-A phosphorylation by P-TEFb is a key event in the pausing release. We conduct IMAC-LC-MS-MS to identified NELF-A phosphorylation site by activation P-TEFb kinase.
Project description:DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain obscure. Here we show that following DNA damage, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) elongation and increases cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by DNA damage-enhanced binding of RBM7 to 7SK, the scaffold of the 7SK small nuclear ribonucleoprotein (snRNP) that inhibits P-TEFb. As a result, P-TEFb relocates from 7SK snRNP to chromatin to activate transcription of key DDR genes and multiple classes of non-coding RNAs. By alleviating the inhibition of P-TEFb, RBM7 thus promotes Pol II elongation to activate a transcriptional response that is crucial for cell fate upon genotoxic insult. Our work highlights a novel paradigm in stress-dependent control of Pol II pause release, and offers the promise of combating cancer by RBM7 and P-TEFb antagonists in combination with established DNA damage-inducing chemotherapeutics.
Project description:Loss of Tsc1 in the kidneys gives rise to rapid polycystogenesis and kidney failure due to the hyperactivation of mTOR complex I (mTORC1). One of the major downstream targets of mTORC1 is S6K1, which is responsible for driving many of the effector functions of mTOR. Strikingly, the loss of S6k1 in the context of hyperactivated mTOR is sufficient to reverse the generation of polycystic kidneys. In an effort to uncover novel direct S6k1 substrates involved in the generation of cystic kidneys, we generated Tsc1-null and Tsc1/S6k1-double null murine inner medullary collecting duct (iMCD3) cell lines using CRISPR/Cas9. Using these two cell lines, we conducted a phosphoproteome screen to search for novel candidates. Using a combination of antibody-motif enrichment and metal affinity enrichment, we provide a comprehensive phosphoproteome profile between these two genotypes.
Project description:Modern omics technologies allow us obtaining global information on different types of biological networks. However, integrating these different types of analyzes into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially-defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS mediated signaling is the strongest from the plasma membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal new HRAS functions including the control of cell migration from the endoplasmic reticulum and p53 dependent cell survival when signaling from the Golgi apparatus.
Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.
Project description:Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase that regulates a plethora of cellular processes. PP2A operates as a holoenzyme complex, comprising one each of the scaffolding (A), regulatory (B) and catalytic (C) subunits. PPP2CA is the principal catalytic subunit of the PP2A holoenzyme complex. Although previous studies have reported many substrates of specific PP2A holoenzyme complexes, the full scope of PP2A substrates in cells remains to be defined. To address this, we generated HEK293 cells in which PPP2CA was homozygously knocked in with a dTAG, allowing for efficient and selective degradation of dTAG-PPP2CA with proteolysis-targeting chimeras (PROTACs) targeting the dTAG. By employing an unbiased global phospho-proteomic analysis, we identified 6,280 phospho-peptides corresponding to 2,204 proteins that showed a significant increase in abundance upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. Among these, some were established PP2A substrates, while most were novel. Bioinformatic analyses revealed the involvement of the identified potential PPP2CA substrates in many cellular processes, including spliceosome function, the cell cycle, RNA transport and ubiquitin-mediated proteolysis. We show that a pSP/pTP motif is a predominant target for PPP2CA. We confirmed some of our phosphoproteomic data with immunoblotting, by utilising commercially available phospho-specific antibodies. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.
Project description:A major driver of multiple myeloma is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employ an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in multiple myeloma to inform precision medicine strategies. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between <I>NRAS</I> and <I>KRAS</I> mutants invisible to the genomic landscape. Transcriptional analysis suggests that aberrant MAPK pathway activation is only present in a fraction of <I>RAS</I>-mutated vs. WT <I>RAS</I> patients. These high-MAPK patients, enriched for <I>NRAS</I> Q61 mutations, have inferior outcomes whereas <I>RAS</I> mutations overall carry no survival impact. We further develop an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. These results may lead to improved stratification of MM patients in clinical trials while also revealing unexplored modes of Ras biology.
Project description:Reversible protein phosphorylation, catalysed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signalling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilised anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR/Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.
Project description:Using Huntington’s disease (HD) mouse models that quantitatively replicate the reduction of striatal Phoshodiesterase 10 (PDE10) levels in manifest Huntington’s disease patients, we demonstrate the potential therapeutic benefit of PDE10 inhibition on correcting basal ganglia circuitry deficits thought to drive disease symptomology in patients. PDE10 inhibition acutely restored corticostriatal input and boosted cortically driven indirect pathway activity in HD models with compromised PDE10 levels. We show that cyclic nucleotide signaling processes are impaired in the models, and that elevation of both the cAMP and cGMP nucleotides afforded by PDE10 inhibition are required for this rescue. Global phosphoproteomic profiling of striatal proteins in response to PDE10 inhibition provide novel information on the plausible neural substrates responsible for the improvement. Finally, we show that long-term chronic treatment of the Q175 knock-in mouse model with PDE10A inhibitors, starting at a presymptomatic age, showed improvements, above and beyond those seen during acute administration, including a partial reversal of striatal deregulated transcripts predominantly driven through activation of the AP-1 and CREB transcription factor complexes, and a prevention of the emergence of some cardinal HD neurophysiological deficits.
Project description:CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9’s activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb’s loss of activity, only the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. ChIP-seq of Pol II in HeLa cells before or after i-CDk9 treatment