Publications by authors named "Michael Wierer"

25 Publications

  • Page 1 of 1

A proteomic atlas of the neointima identifies novel druggable targets for preventive therapy.

Eur Heart J 2021 Apr 8. Epub 2021 Apr 8.

Department of Cardiology, German Heart Centre Munich, Technical University of Munich, Munich, Germany.

Aims : In-stent restenosis is a complication after coronary stenting associated with morbidity and mortality. Here, we sought to investigate the molecular processes underlying neointima formation and to identify new treatment and prevention targets.

Methods And Results : Neointima formation was induced by wire injury in mouse femoral arteries. High-accuracy proteomic measurement of single femoral arteries to a depth of about 5000 proteins revealed massive proteome remodelling, with more than half of all proteins exhibiting expression differences between injured and non-injured vessels. We observed major changes in the composition of the extracellular matrix and cell migration processes. Among the latter, we identified the classical transient receptor potential channel 6 (TRPC6) to drive neointima formation. While Trpc6-/- mice presented reduced neointima formation compared to wild-type mice (1.44 ± 0.39 vs. 2.16 ± 0.48, P = 0.01), activating or repressing TRPC6 in human vascular smooth muscle cells resulted in increased [vehicle 156.9 ± 15.8 vs. 1-oleoyl-2-acetyl-sn-glycerol 179.1 ± 8.07 (103 pixels), P = 0.01] or decreased migratory capacity [vehicle 130.0 ± 26.1 vs. SAR7334 111.4 ± 38.0 (103 pixels), P = 0.04], respectively. In a cohort of individuals with angiographic follow-up (n = 3068, males: 69.9%, age: 59 ± 11 years, follow-up 217.1 ± 156.4 days), homozygous carriers of a common genetic variant associated with elevated TRPC6 expression were at increased risk of restenosis after coronary stenting (adjusted odds ratio 1.49, 95% confidence interval 1.08-2.05; P = 0.01).

Conclusions : Our study provides a proteomic atlas of the healthy and injured arterial wall that can be used to define novel factors for therapeutic targeting. We present TRPC6 as an actionable target to prevent neointima formation secondary to vascular injury and stent implantation.
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http://dx.doi.org/10.1093/eurheartj/ehab140DOI Listing
April 2021

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.

Nucleic Acids Res 2021 Mar 3. Epub 2021 Mar 3.

Department of Biology II and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
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http://dx.doi.org/10.1093/nar/gkab132DOI Listing
March 2021

The Transcription Factor MAFF Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

Circulation 2021 Feb 25. Epub 2021 Feb 25.

Department of Cardiology, Deutsches Herzzentrum München, Technische Universität München, Munich, Germany; Deutsches Zentrum für Herz- und Kreislauferkrankungen (DZHK), Partner Site Munich Heart Alliance, Munich, Germany.

Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterise central regulators and networks leading to atherosclerosis. Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knock-out models) and human (as shown by genome-wide association studies (GWAS)) liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models as well as experimental studies including chromatin immunoprecipitation DNA-Sequencing (ChIP-Seq), ChIP mass spectrometry (ChIP-MS), overexpression, siRNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. The transcription factor interacted as a key driver of a liver network with three human genes at CAD GWAS loci and eleven atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor () gene correlated with in 600 CAD patients undergoing bypass surgery (STARNET) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of were tested under non-inflammatory conditions showing a positive correlation between and and . Interestingly, after LPS stimulation (inflammatory conditions) an inverse correlation between and and was observed. ChIP-MS revealed that the human CAD GWAS candidate assists in the presence of LPS stimulation with respective heterodimers binding at the MAF recognition element (MARE) of the promoter to transcriptionally downregulate expression. The transcription factor was identified as a novel central regulator of an atherosclerosis/CAD relevant liver network. triggered context specific expression of and other genes known to affect CAD risk. Our results suggest that is a missing link between inflammation, lipid and lipoprotein metabolism and a possible treatment target.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.050186DOI Listing
February 2021

The glucocorticoid receptor recruits the COMPASS complex to regulate inflammatory transcription at macrophage enhancers.

Cell Rep 2021 Feb;34(6):108742

Institute for Diabetes and Obesity (IDO) & Institute for Diabetes and Cancer (IDC), Helmholtz Center Munich (HMGU) and German Center for Diabetes Research (DZD), 85764 Neuherberg (Munich), Germany; Metabolic Programming, School of Life Sciences Weihenstephan, ZIEL - Institute for Food & Health, Technische Universitaet Muenchen (TUM), 85354 Freising, Germany; Metabolic Biochemistry and Genetics, Gene Center, Ludwig-Maximilians-Universitaet LMU, 81377 Munich, Germany. Electronic address:

Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis-regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.
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http://dx.doi.org/10.1016/j.celrep.2021.108742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873837PMC
February 2021

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
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http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

Anti-inflammatory functions of the glucocorticoid receptor require DNA binding.

Nucleic Acids Res 2020 09;48(15):8393-8407

Molecular Endocrinology, Institutes for Diabetes and Obesity & Diabetes and Cancer IDO & IDC, Helmholtz Zentrum Muenchen (HMGU) and German Center for Diabetes Research (DZD), Munich 85764, Germany.

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.
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http://dx.doi.org/10.1093/nar/gkaa565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470971PMC
September 2020

Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry.

Nat Commun 2020 06 15;11(1):3019. Epub 2020 Jun 15.

Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.

Transcription factors (TFs) regulate target genes by specific interactions with DNA sequences. Detecting and understanding these interactions at the molecular level is of fundamental importance in biological and clinical contexts. Crosslinking mass spectrometry is a powerful tool to assist the structure prediction of protein complexes but has been limited to the study of protein-protein and protein-RNA interactions. Here, we present a femtosecond laser-induced crosslinking mass spectrometry (fliX-MS) workflow, which allows the mapping of protein-DNA contacts at single nucleotide and up to single amino acid resolution. Applied to recombinant histone octamers, NF1, and TBP in complex with DNA, our method is highly specific for the mapping of DNA binding domains. Identified crosslinks are in close agreement with previous biochemical data on DNA binding and mostly fit known complex structures. Applying fliX-MS to cells identifies several bona fide crosslinks on DNA binding domains, paving the way for future large scale ex vivo experiments.
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http://dx.doi.org/10.1038/s41467-020-16837-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295792PMC
June 2020

Kreuth V initiative: European consensus proposals for treatment of hemophilia using standard products, extended half-life coagulation factor concentrates and non-replacement therapies.

Haematologica 2020 08 28;105(8):2038-2043. Epub 2020 May 28.

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Milan, Italy.

This report contains the updated consensus recommendations for optimal hemophilia care produced in 2019 by three Working Groups (WG) on behalf of the European Directorate for Quality of Medicines and Healthcare in the frame of the Kreuth V Initiative. WG1 recommended access to prophylaxis for all patients, the achievement of plasma factor trough levels of at least 3-5% when extended half-life factor VIII (FVIII) and FIX products are used, a personalized treatment regimen, and a choice of chromogenic assays for treatment monitoring. It was also emphasized that innovative therapies should be supervised by hemophilia comprehensive care centers. WG2 recommended mandatory collection of postmarketing data to assure the long-term safety and efficacy of new hemophilia therapies, the establishment of national patient registries including the core data recommended by the European Medicines Agency and the International Society on Thrombosis and Haemostasis, with adequate support under public control, and greater collaboration to facilitate a comprehensive data evaluation throughout Europe. WG3 discussed methodological aspects of hemophilia care in the context of access decisions, particularly for innovative therapies, and recommended that clinical studies should be designed to provide the quality of evidence needed by regulatory authorities, HTA bodies and healthcare providers. The dialogue between all stakeholders in hemophilia care and patient organizations should be fostered to implement these recommendations.
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http://dx.doi.org/10.3324/haematol.2019.242735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395279PMC
August 2020

SWR1 and NuA4 complexes are defined by DOMINO isoforms.

Elife 2020 05 20;9. Epub 2020 May 20.

Molecular Biology Division, Biomedical Center, Ludwig-Maximilians-University, Munich, Germany.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In , the single DOMINO chromatin regulator complex is thought to combine both activities an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. generates the same diversity by alternative splicing of a single gene.
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http://dx.doi.org/10.7554/eLife.56325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239659PMC
May 2020

Cistromic Reprogramming of the Diurnal Glucocorticoid Hormone Response by High-Fat Diet.

Mol Cell 2019 11 6;76(4):531-545.e5. Epub 2019 Nov 6.

Institute for Diabetes and Obesity (IDO), Helmholtz Center Munich (HMGU) and German Center for Diabetes Research (DZD), Ingolstaedter Landstr. 1, 85764 Neuherberg (Munich), Germany; Institute for Diabetes and Cancer (IDC), Helmholtz Center Munich (HMGU) and German Center for Diabetes Research (DZD), Ingolstaedter Landstr. 1, 85764 Neuherberg (Munich), Germany; Metabolic Programming, School of Life Sciences Weihenstephan, Gregor Mendel Str. 2, 85354 Freising, Technische Universitaet Muenchen (TUM), Munich, Germany. Electronic address:

The glucocorticoid receptor (GR) is a potent metabolic regulator and a major drug target. While GR is known to play integral roles in circadian biology, its rhythmic genomic actions have never been characterized. Here we mapped GR's chromatin occupancy in mouse livers throughout the day and night cycle. We show how GR partitions metabolic processes by time-dependent target gene regulation and controls circulating glucose and triglycerides differentially during feeding and fasting. Highlighting the dominant role GR plays in synchronizing circadian amplitudes, we find that the majority of oscillating genes are bound by and depend on GR. This rhythmic pattern is altered by high-fat diet in a ligand-independent manner. We find that the remodeling of oscillatory gene expression and postprandial GR binding results from a concomitant increase of STAT5 co-occupancy in obese mice. Altogether, our findings highlight GR's fundamental role in the rhythmic orchestration of hepatic metabolism.
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http://dx.doi.org/10.1016/j.molcel.2019.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928064PMC
November 2019

Functional identity of hypothalamic melanocortin neurons depends on Tbx3.

Nat Metab 2019 02 28;1(2):222-235. Epub 2019 Jan 28.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany.

Heterogeneous populations of hypothalamic neurons orchestrate energy balance via the release of specific signatures of neuropeptides. However, how specific intracellular machinery controls peptidergic identities and function of individual hypothalamic neurons remains largely unknown. The transcription factor T-box 3 (Tbx3) is expressed in hypothalamic neurons sensing and governing energy status, whereas human TBX3 haploinsufficiency has been linked with obesity. Here, we demonstrate that loss of Tbx3 function in hypothalamic neurons causes weight gain and other metabolic disturbances by disrupting both the peptidergic identity and plasticity of Pomc/Cart and Agrp/Npy neurons. These alterations are observed after loss of Tbx3 in both immature hypothalamic neurons and terminally differentiated mouse neurons. We further establish the importance of Tbx3 for body weight regulation in Drosophila melanogaster and show that TBX3 is implicated in the differentiation of human embryonic stem cells into hypothalamic Pomc neurons. Our data indicate that Tbx3 directs the terminal specification of neurons as functional components of the melanocortin system and is required for maintaining their peptidergic identity. In summary, we report the discovery of a key mechanistic process underlying the functional heterogeneity of hypothalamic neurons governing body weight and systemic metabolism.
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http://dx.doi.org/10.1038/s42255-018-0028-1DOI Listing
February 2019

E47 modulates hepatic glucocorticoid action.

Nat Commun 2019 01 18;10(1):306. Epub 2019 Jan 18.

Molecular Endocrinology, Helmholtz Diabetes Center (HMGU) and German Center for Diabetes Research (DZD), IDO, Ingolstädter Landstr. 1, 85764, Neuherberg, Munich, Germany.

Glucocorticoids (GCs) are effective drugs, but their clinical use is compromised by severe side effects including hyperglycemia, hyperlipidemia and obesity. They bind to the Glucocorticoid Receptor (GR), which acts as a transcription factor. The activation of metabolic genes by GR is thought to underlie these adverse effects. We identify the bHLH factor E47 as a modulator of GR target genes. Using mouse genetics, we find that E47 is required for the regulation of hepatic glucose and lipid metabolism by GR, and that loss of E47 prevents the development of hyperglycemia and hepatic steatosis in response to GCs. Here we show that E47 and GR co-occupy metabolic promoters and enhancers. E47 is needed for the efficient recruitment of GR and coregulators such as Mediator to chromatin. Altogether, our results illustrate how GR and E47 regulate hepatic metabolism, and might provide an entry point for novel therapies with reduced side effects.
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http://dx.doi.org/10.1038/s41467-018-08196-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338785PMC
January 2019

Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer.

Cell 2018 09;175(1):159-170.e16

Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, University of Chicago, Chicago, IL 60637, USA.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.
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http://dx.doi.org/10.1016/j.cell.2018.08.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827878PMC
September 2018

Rapid proteomic analysis for solid tumors reveals LSD1 as a drug target in an end-stage cancer patient.

Mol Oncol 2018 08 14;12(8):1296-1307. Epub 2018 Jun 14.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

Recent advances in mass spectrometry (MS)-based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine-specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can be applied broadly to other patients in a similar condition.
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http://dx.doi.org/10.1002/1878-0261.12326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068348PMC
August 2018

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

Mol Cell Proteomics 2018 02 4;17(2):321-334. Epub 2017 Dec 4.

From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany;

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function.
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http://dx.doi.org/10.1074/mcp.RA117.000315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795394PMC
February 2018

EPOP Functionally Links Elongin and Polycomb in Pluripotent Stem Cells.

Mol Cell 2016 11;64(4):645-658

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain; ICREA, Pg. Lluis Companys 23, 08010 Barcelona, Spain. Electronic address:

The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.
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http://dx.doi.org/10.1016/j.molcel.2016.10.018DOI Listing
November 2016

Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes.

Hum Mol Genet 2016 Oct 11;25(R2):R106-R114. Epub 2016 Jul 11.

Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany

High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation - either isotope-based or label free - unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics.
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http://dx.doi.org/10.1093/hmg/ddw208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036873PMC
October 2016

Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

Basic Res Cardiol 2016 07 24;111(4):51. Epub 2016 Jun 24.

Klinik für Herz- und Kreislauferkrankungen, Deutsches Herzzentrum München, Technische Universität München, Lazarettstr. 36, 80636, Munich, Germany.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.
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http://dx.doi.org/10.1007/s00395-016-0570-5DOI Listing
July 2016

ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

Science 2016 Jun;352(6290):1221-5

Centre de Regulació Genòmica (CRG), Barcelona Institute for Science and Technology, Barcelona E-09003, Spain. Universitat Pompeu Fabra, Barcelona E-08003, Spain.

Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.
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http://dx.doi.org/10.1126/science.aad9335DOI Listing
June 2016

C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4.

Nat Cell Biol 2016 Apr 14;18(4):371-81. Epub 2016 Mar 14.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPα (Bα' cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPα also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Bα' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPα.
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http://dx.doi.org/10.1038/ncb3326DOI Listing
April 2016

Quality standards of the European Pharmacopoeia.

J Ethnopharmacol 2014 Dec 17;158 Pt B:454-7. Epub 2014 Jul 17.

European Directorate for the Quality of Medicines & Healthcare (EDQM), Council of Europe, 7 Allée Kastner, Cs 30026, F-67081 Strasbourg, France.

Ethnopharmacological Relevance: The European Pharmacopoeia (Ph. Eur.) provides a legal and scientific reference for the quality control of medicines. It is legally binding in the 38 signatory parties of the Convention on the elaboration of a European Pharmacopoeia (37 member states and the European Union).

Materials And Methods: The requirements for a specific herbal drug are prescribed in the corresponding individual monograph and the relevant general monographs. Criteria for pesticides and heavy metals for example are defined in the general monograph on Herbal drugs. The Ph. Eur. also provides general methods including methods for determination of aflatoxins B1 and ochratoxin A. Screening methods for aristolochic acids are applied for herbal drugs that may be subject to adulteration or substitution with plant material containing aristolochic acids.

Conclusion: The Ph. Eur. collaborate in many areas with the European Medicines Agency (EMA) to ensure close collaboration as regards the respective work programmes and approach.
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http://dx.doi.org/10.1016/j.jep.2014.07.020DOI Listing
December 2014

PLK1 signaling in breast cancer cells cooperates with estrogen receptor-dependent gene transcription.

Cell Rep 2013 Jun 13;3(6):2021-32. Epub 2013 Jun 13.

Gene Regulation Stem Cells and Cancer Program, Center for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.

Polo-like kinase 1 (PLK1) is a key regulator of cell division and is overexpressed in many types of human cancers. Compared to its well-characterized role in mitosis, little is known about PLK1 functions in interphase. Here, we report that PLK1 mediates estrogen receptor (ER)-regulated gene transcription in human breast cancer cells. PLK1 interacts with ER and is recruited to ER cis-elements on chromatin. PLK1-coactivated genes included classical ER target genes such as Ps2, Wisp2, and Serpina3 and were enriched in developmental and tumor-suppressive functions. Performing large-scale phosphoproteomics of estradiol-treated MCF7 cells in the presence or absence of the specific PLK1 inhibitor BI2536, we identified several PLK1 end targets involved in transcription, including the histone H3K4 trimethylase MLL2, the function of which on ER target genes was impaired by PLK1 inhibition. Our results propose a mechanism for the tumor-suppressive role of PLK1 in mammals as an interphase transcriptional regulator.
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http://dx.doi.org/10.1016/j.celrep.2013.05.024DOI Listing
June 2013

A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

PLoS One 2012 27;7(11):e50350. Epub 2012 Nov 27.

Physiology Weihenstephan, Technical University Munich, Freising, Germany.

The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050350PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507690PMC
June 2013

Data extraction from proteomics raw data: an evaluation of nine tandem MS tools using a large Orbitrap data set.

J Proteomics 2012 Sep 20;75(17):5293-303. Epub 2012 Jun 20.

Centro de Regulación Genòmica (CRG), C/Dr. Aiguader 88, 08003 Barcelona, Spain.

In shot-gun proteomics raw tandem MS data are processed with extraction tools to produce condensed peak lists that can be uploaded to database search engines. Many extraction tools are available but to our knowledge, a systematic comparison of such tools has not yet been carried out. Using raw data containing more than 400,000 tandem MS spectra acquired using an Orbitrap Velos we compared 9 tandem MS extraction tools, freely available as well as commercial. We compared the tools with respect to number of extracted MS/MS events, fragment ion information, number of matches, precursor mass accuracies and agreement in-between tools. Processing a primary data set with 9 different tandem MS extraction tools resulted in a low overlap of identified peptides. The tools differ by assigned charge states of precursors, precursor and fragment ion masses, and we show that peptides identified very confidently using one extraction tool might not be matched when using another tool. We also found a bias towards peptides of lower charge state when extracting fragment ion data from higher resolution raw data without deconvolution. Collecting and comparing the extracted data from the same raw data allow adjusting parameters and expectations and selecting the right tool for extraction of tandem MS data.
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http://dx.doi.org/10.1016/j.jprot.2012.06.012DOI Listing
September 2012