Publications by authors named "Chunaram Choudhary"

80 Publications

Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

Mol Cell 2021 Mar 17. Epub 2021 Mar 17.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen 2200, Denmark. Electronic address:

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2021.03.008DOI Listing
March 2021

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Mol Cell 2021 03 14;81(5):1084-1099.e6. Epub 2021 Jan 14.

The Novo Nordisk Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939521PMC
March 2021

Equilibrium between nascent and parental MCM proteins protects replicating genomes.

Nature 2020 11 21;587(7833):297-302. Epub 2020 Oct 21.

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2842-3DOI Listing
November 2020

Histone Acetyltransferase MOF Blocks Acquisition of Quiescence in Ground-State ESCs through Activating Fatty Acid Oxidation.

Cell Stem Cell 2020 09 30;27(3):441-458.e10. Epub 2020 Jun 30.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address:

Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758074PMC
September 2020

Sequencing of the First Draft of the Human Acetylome.

Clin Chem 2020 06;66(6):852-853

Proteomics Program, the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/clinchem/hvaa087DOI Listing
June 2020

HBO1 is required for the maintenance of leukaemia stem cells.

Nature 2020 01 11;577(7789):266-270. Epub 2019 Dec 11.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs). Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1835-6DOI Listing
January 2020

Analysis and Interpretation of Protein Post-Translational Modification Site Stoichiometry.

Trends Biochem Sci 2019 11 8;44(11):943-960. Epub 2019 Jul 8.

Proteomics Program, the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. Electronic address:

Proteins are decorated with a diverse array of post-translational modifications (PTMs) that regulate their spatial and temporal functions. Recent mass spectrometry (MS)-based studies have identified hundreds of thousands of PTM sites in mammalian proteomes. However, the signaling cues and enzymes regulating individual sites are often not known and their functional roles remain uncharacterized. Quantification of PTM site stoichiometry can help in prioritizing sites for functional analyses and is important for constructing mechanistic models of PTM-dependent protein regulation. Here, we review the concept of PTM site stoichiometry, critically evaluate the merits and drawbacks of different MS-based methods used for quantifying PTM site stoichiometry, and discuss the usefulness and limitations of stoichiometry in informing on the biological function of modified sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tibs.2019.06.003DOI Listing
November 2019

Author Correction: Functions and mechanisms of non-histone protein acetylation.

Nat Rev Mol Cell Biol 2019 Aug;20(8):508

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41580-019-0156-9DOI Listing
August 2019

Retraction.

Science 2019 04 11;364(6437):247. Epub 2019 Apr 11.

The Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aax4558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558281PMC
April 2019

Analysis of human acetylation stoichiometry defines mechanistic constraints on protein regulation.

Nat Commun 2019 03 5;10(1):1055. Epub 2019 Mar 5.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200, Copenhagen, Denmark.

Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. However, the stoichiometry of acetylation remains poorly characterized, and is important for understanding acetylation-dependent mechanisms of protein regulation. Here we provide accurate, validated measurements of acetylation stoichiometry at 6829 sites on 2535 proteins in human cervical cancer (HeLa) cells. Most acetylation occurs at very low stoichiometry (median 0.02%), whereas high stoichiometry acetylation (>1%) occurs on nuclear proteins involved in gene transcription and on acetyltransferases. Analysis of acetylation copy numbers show that histones harbor the majority of acetylated lysine residues in human cells. Class I deacetylases target a greater proportion of high stoichiometry acetylation compared to SIRT1 and HDAC6. The acetyltransferases CBP and p300 catalyze a majority (65%) of high stoichiometry acetylation. This resource dataset provides valuable information for evaluating the impact of individual acetylation sites on protein function and for building accurate mechanistic models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09024-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401094PMC
March 2019

Acetylation of intrinsically disordered regions regulates phase separation.

Nat Chem Biol 2019 01 10;15(1):51-61. Epub 2018 Dec 10.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Liquid-liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-018-0180-7DOI Listing
January 2019

Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint.

Cell Rep 2018 11;25(9):2317-2328.e5

Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The multisubunit ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. APC/C is tightly regulated by the spindle assembly checkpoint (SAC), which involves MPS1 and MAD2-dependent temporal inhibition of APC/C. We analyzed the contribution of the APC/C subunits APC7 and APC16 to APC/C composition and function in human cells. APC16 is required for APC7 assembly into APC/C, whereas APC16 assembles independently of APC7. APC7 and APC16 knockout cells display no major defects in mitotic progression, cyclin B1 degradation, or SAC response, but APC/C lacking these two subunits shows reduced ubiquitylation activity in vitro. Strikingly, deletion of APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. ΔAPC7ΔMAD2 cells display accelerated mitosis and require SAC-independent MPS1 function for genome stability. These findings reveal that the composition of APC/C critically influences the importance of the SAC in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.10.104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289045PMC
November 2018

Functions and mechanisms of non-histone protein acetylation.

Nat Rev Mol Cell Biol 2019 03;20(3):156-174

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Nε-lysine acetylation was discovered more than half a century ago as a post-translational modification of histones and has been extensively studied in the context of transcription regulation. In the past decade, proteomic analyses have revealed that non-histone proteins are frequently acetylated and constitute a major portion of the acetylome in mammalian cells. Indeed, non-histone protein acetylation is involved in key cellular processes relevant to physiology and disease, such as gene transcription, DNA damage repair, cell division, signal transduction, protein folding, autophagy and metabolism. Acetylation affects protein functions through diverse mechanisms, including by regulating protein stability, enzymatic activity, subcellular localization and crosstalk with other post-translational modifications and by controlling protein-protein and protein-DNA interactions. In this Review, we discuss recent progress in our understanding of the scope, functional diversity and mechanisms of non-histone protein acetylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41580-018-0081-3DOI Listing
March 2019

STK3 is a therapeutic target for a subset of acute myeloid leukemias.

Oncotarget 2018 May 22;9(39):25458-25473. Epub 2018 May 22.

Universitäts KrebsCentrums Dresden, Medical Systems Biology, Medical Faculty, Technische Universität Dresden, Dresden, Germany.

Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.25238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986655PMC
May 2018

Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.

Cell 2018 06 24;174(1):231-244.e12. Epub 2018 May 24.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078418PMC
June 2018

Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

Nature 2018 06;558(7710):E1

Discovery, Global Pharmaceutical Research and Development, North Chicago, IL, USA.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0111-5DOI Listing
June 2018

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Cell 2018 05 12;173(4):972-988.e23. Epub 2018 Apr 12.

Proteomics Program, the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark; Center for Chromosome Stability (CCS), Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. Electronic address:

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.03.050DOI Listing
May 2018

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Nat Commun 2018 03 9;9(1):1017. Epub 2018 Mar 9.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-03417-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845016PMC
March 2018

lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1.

Cell Rep 2017 Dec;21(11):3012-3021

Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; German Cancer Research Center (DKFZ), Heidelberg and German Cancer Consortium (DKTK) partner site Dresden, 01307 Dresden, Germany; National Center for Tumor Diseases (NCT), University Hospital Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany. Electronic address:

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.11.045DOI Listing
December 2017

Redox-sensitive alteration of replisome architecture safeguards genome integrity.

Science 2017 11;358(6364):797-802

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aao3172DOI Listing
November 2017

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

Nature 2017 10 27;550(7674):128-132. Epub 2017 Sep 27.

Discovery, Global Pharmaceutical Research and Development, AbbVie, 1 North Waukegan Road, North Chicago, Illinois 60064, USA.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature24028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050590PMC
October 2017

Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on the Acetylome.

Mol Cell Proteomics 2017 05 2;16(5):759-769. Epub 2017 Mar 2.

From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylated peptides to native acetylated peptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (Δ) and acetate kinase (Δ) mutant strains in the presence and absence of the Sirtuin inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such as stationary-phase arrest and deletion of , resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry in WT, Δ, and Δ cells. Together, our results provide a detailed view of acetylation stoichiometry in and suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.M117.067587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417819PMC
May 2017

Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover.

Cell Discov 2016 29;2:16001. Epub 2016 Mar 29.

Department of Cellular and Molecular Medicine, University of Copenhagen , Copenhagen, Denmark.

The Hippo pathway has been identified as a key barrier for tumorigenesis, acting through downregulation of YAP/TAZ activity. Elevated YAP/TAZ activity has been documented in many human cancers. Ubiquitylation has been shown to play a key role in regulating YAP/TAZ activity through downregulation of a number of Hippo pathway components. Several ubiquitin ligase complexes have been implicated in this process, however, little is known about the deubiquitylating enzymes that counteract these activities to regulate YAP/TAZ. Here we identify the deubiquitylating enzyme USP9x as a regulator of YAP/TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x mRNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/celldisc.2016.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849470PMC
July 2016

SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes.

EMBO J 2016 09 15;35(17):1868-84. Epub 2016 Jun 15.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

TNF-α is a key regulator of innate immune and proinflammatory responses. However, the composition of the TNF-α receptor-associated signaling complexes (TNF-RSC) and the architecture of the downstream signaling networks are incompletely understood. We employed quantitative mass spectrometry to demonstrate that TNF-α stimulation induces widespread protein phosphorylation and that the scope of phosphorylation expands in a temporal manner. TNF-α stimulation also induces rapid ubiquitylation of components of the TNF-RSC Temporal analysis of the TNF-RSC composition identified SPATA2 as a novel component of the TNF-RSC The predicted PUB domain in the N-terminus of SPATA2 interacts with the USP domain of CYLD, whereas the C-terminus of SPATA2 interacts with HOIP SPATA2 is required for recruitment of CYLD to the TNF-RSC Downregulation of SPATA2 augments transcriptional activation of NF-κB and inhibits TNF-α-induced necroptosis, pointing to an important function of SPATA2 in modulating the outcomes of TNF-α signaling. Taken together, our study draws a detailed map of TNF-α signaling, identifies SPATA2 as a novel component of TNF-α signaling, and provides a rich resource for further functional investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201694300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007551PMC
September 2016

Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells.

Cell Syst 2015 Aug 26;1(2):141-51. Epub 2015 Aug 26.

Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. Electronic address:

We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a framework for better understanding pluripotency and for dissecting the molecular events that govern the transition from the pre-implantation to the post-implantation state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2015.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023010PMC
August 2015

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance.

Genes Dev 2016 Mar 10;30(6):700-17. Epub 2016 Mar 10.

Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark.

Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.276204.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803055PMC
March 2016

The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.

Cell Rep 2016 Mar 18;14(8):1829-40. Epub 2016 Feb 18.

Proteomics Program, the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.01.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785794PMC
March 2016

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Genes Dev 2016 Jan 7;30(2):149-63. Epub 2016 Jan 7.

Chromatin Structure and Function Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark;

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.268797.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719306PMC
January 2016

Avoiding abundance bias in the functional annotation of post-translationally modified proteins.

Nat Methods 2015 Nov;12(11):1003-4

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3621DOI Listing
November 2015

Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage.

Nature 2015 Nov 21;527(7578):389-93. Epub 2015 Oct 21.

Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature15401DOI Listing
November 2015