Molecular Mechanisms of Chromatin Regulation by DNA and Histone Modifications and Epigenetic Reader Molecules

Lead Research Organisation: MRC Clinical Sciences Centre

Abstract

The instructions for building a complete organism are stored on long DNA molecules called chromosomes in every cell of our body. The information is encoded in the DNA sequences of genes which can be either active or inactive depending on the type of cell. How does a particular type of cell switch on one set of genes while a different type activates another? It has become apparent that the way a gene is packaged by proteins is very important in determining its activity. Genes are packaged by histone proteins and these histones, and also the DNA itself, can be modified by the addition of chemical groups. These chemical modifications can in turn attract other proteins that can read this “epigenetic” information contained in the modifications and thus influence the activity of a gene. The distribution of DNA and histone modifications across the chromosomes is abnormal in many diseases, in particular in cancer. We, therefore, want to understand the “language” of these modifications. Using biochemical and mass-spectrometric experimental techniques we aim to identify the proteins that can recognise and interpret the information encoded in DNA and histone modifications. Our goal is to use this knowledge in order to develop drugs that can revert abnormal DNA and histone modifications in so called epigenetic therapies.

Technical Summary

Most of the genetic information in eukaryotic cells is stored within the nucleus in the form of chromatin. Two key mechanisms known to regulate the functional state of chromatin in mammals are the methylation of DNA and the post-translational modification of histone proteins. Due to the large number of possible modifications, epigenetic information can be stored in chromatin modification patterns.
Chromatin modifications have been shown to regulate all DNA-associated processes, such as transcription, replication, and DNA repair, and play an important role in cell proliferation and differentiation. These functions are intimately linked to the faithful interpretation and inheritance of genetic information and the memory of a cell’s identity. Deregulation of these modifications and their modifying enzymes are implicated in many types of diseases, including cancer and neurological disorders.
Many chromatin-regulating factors have been identified that recognise methylated DNA or modified histones. Such effector molecules use a range of different binding domains in order to establish and orchestrate biological events. Since chromatin is a large macromolecular assembly, modifications most likely act in a concerted manner. However, it is still unclear how the information contained in combinatorial modification patterns on the DNA and histones is interpreted.
Our aim is to understand how combinations of DNA and histone modifications regulate the activity of chromatin. We employ the tools of chemical biology, biochemistry and proteomics in conjunction with tissue culture and genomic technologies in order to study proteins that can recognise DNA and histone modification patterns in the context of chromatin.
We have developed a technique called SILAC nucleosome affinity purification that allows us to identify proteins that bind DNA or histone modifications on in vitro assembled nucleosomes using high-resolution mass spectrometry. We will use this technique for identifying new factors that integrate information contained in multiple chromatin modifications on nucleosomes and chromatin, and for understanding how they operate at the molecular level. We are particularly interested in the molecular mechanisms that underlie epigenetic gene regulation events during DNA replication, tumour formation and differentiation processes.
Our research will help elucidate how chromatin modifications regulate cellular processes and how deregulation of normal chromatin function leads to diseases. Our ultimate goal is to identify the critical factors and to understand their molecular and cellular functions in order to develop drugs for epigenetic therapies against diseases such as cancer.
 
Description BBSRC Doctoral Training Programme
Amount £84,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2013 
End 09/2017
 
Description BBSRC Responsive Mode Research Grant
Amount £11,926 (GBP)
Funding ID P50645 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2014 
End 10/2017
 
Description ERC Starting Grant
Amount € 1,630,000 (EUR)
Funding ID 309952/ROCCOMO 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 05/2013 
End 04/2018
 
Description ICCS - Interdisciplinary, Cross-Campus, Collaborative Studentship
Amount £72,000 (GBP)
Funding ID ICCS 
Organisation Imperial College London (ICL) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2013 
End 03/2017
 
Title Library of post-translationally modified histones and nucleosomes 
Description Using the native chemical ligation technique we have generated around 30 different variants of histones H3 and H4 carrying a diverse set of post-translational modifications. These histones have been refolded into ~60 different histone octamers carrying combinations of histone modifications that allow the reconstitution of different types of nucleosomes resembling e.g. promoters or enhancers (in active or inactive states) and different types of heterochromatin. These histones and nucleosomes are available to the community as a resource and will be provided on a collaborative basis upon request. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact We have provided reagents to several labs resulting in a number of publications and a number of manuscripts from current collaborations are under review. Published studies using our reagents are: > Watson AA, Mahajan P, Mertens HD, Deery MJ, Zhang W, Pham P, Du X, Bartke T, Zhang W, Edlich C, Berridge G, Chen Y, Burgess-Brown NA, Kouzarides T, Wiechens N, Owen-Hughes T, Svergund DI, Gileadi O, and Laue ED. (2012). The PHD and chromo-domains regulate the ATPase activity of the human chromatin remodeler CHD4. J. Mol. Biol. 422, 3-17. > Frangini A, Sjöberg M, Roman-Trufero M, Dharmalingam G, Haberle V, Bartke T, Lenhard B, Malumbres M, Vidal M, and Dillon D, (2013). The Aurora B Kinase and the Polycomb Protein Ring1B Combine to Regulate Active Promoters in Quiescent Lymphocytes. Mol. Cell 51, 647-661. > McBurney KL, Leung A, Choi JK, Martin BJE, Irwin NAT, Bartke T, Nelson CJ, and Howe LAJ. (2016). Divergent residues within histone H3 dictate a unique chromatin structure in S. cerevisiae. Genetics 202, 341-349. DOI: 10.1534/genetics.115.180810. Epub 2015 Nov 3. 
 
Description 4DCellfate Young Investigator Programme 
Organisation EC FP7 Collaborative projects
Department 4DCellFate Network
Country Spain, Kingdom of 
Sector Public 
PI Contribution We are providing reagents (modified nucleosomes) to collaboration partners within the network
Collaborator Contribution 1. We can participate in the network conferences (travel and accommodation will be payed for) 2. We have access to structural biology facilities via our collaboration partners within the network
Impact Watson AA, Mahajan P, Mertens HD, Deery MJ, Zhang W, Pham P, Du X, Bartke T, Zhang W, Edlich C, Berridge G, Chen Y, Burgess-Brown NA, Kouzarides T, Wiechens N, Owen-Hughes T, Svergund DI, Gileadi O, and Laue ED. (2012). The PHD and chromo-domains regulate the ATPase activity of the human chromatin remodeler CHD4. J. Mol. Biol. 422, 3-17.
Start Year 2012
 
Description Collaboration with Anja Groth, BRIC/University of Copenhagen 
Organisation University of Copenhagen
Department Faculty of Science
Country Denmark, Kingdom of 
Sector Academic/University 
PI Contribution We have provided reagents for nucleosome interaction studies between the DNA repair factor TONSL and chromatin
Collaborator Contribution Our collaborators carried out the nucleosome interaction studies
Impact This collaboration has resulted in the publication "Saredi G, Huang H, Hammond CM, Alabert C, Bekker-Jensen S, Forne I, Reveron-Gomez N, Foster BM, Mlejnkova L, Bartke T, Cejka P, Mailand N, Imhof A, Patel D, and Groth A. (2016). H4 K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22L DNA repair complex. Nature 534, 714-718. DOI: 10.1038/nature18312."
Start Year 2014
 
Description Collaboration with Elisabeth Busch-Nentwich, Wellcome Trust Sanger Institute 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution Provision of materials and reagents for experiments and intellectual contribution to collaborative project with Elisabeth Busch-Nentwich's group on the function of KDM2A in zebrafish.
Collaborator Contribution Our partners have carried out overexpression experiments of KDM2A mutants in zebrafish embryos for our collaborative project on the function of KDM2A.
Impact This collaboration has resulted in the publication "Borgel J, Tyl M, Schiller K, Pusztai Z, Dooley CM, Deng W, Wooding C, White RJ, Warnecke T, Leonhardt H, Busch-Nentwich E, and Bartke T. (2016). KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1. Nucleic Acids Research. DOI: 10.1093/nar/gkw979."
Start Year 2012
 
Description Collaboration with Epinova/GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are using materials provided by GSK/Epinova to investigate how BRD proteins (Bromodomain containing proteins BRD2 and BRD4) interact with modified chromatin. We are providing experimental chromatin biochemistry and Cell Biological tools and reagents that are used in Peter DiMaggio's lab to identify chromatin modifications that are recognised by BRD2 and BRD4 by mass spectrometric and computational approaches.
Collaborator Contribution GSK/Epinova have provided us with reagents to investigate the impact of Bromodomain Inhibitors on the interaction of BRD proteins with modified chromatin. Peter DiMaggio's lab provides expertise in mass spectrometry and computational approaches that allow the identification of chromatin modifications that are recognised by BRD2 and BRD4.
Impact This is a highly interdisciplinary project combining the expertise in small molecule applications of a large Pharmaceutical Company (GSK) with our expertise in Chromatin Biochemistry and Peter DiMaggio's expertise in mass spectrometry and computational methods. Our current output is the high level interdisciplinary training of a PhD student who is involved in a project combining Cell Biology, Biochemistry, Mass Spectrometry and computational approaches.
Start Year 2015
 
Description Collaboration with Epinova/GSK 
Organisation Imperial College London (ICL)
Department Department of Chemical Engineering
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We are using materials provided by GSK/Epinova to investigate how BRD proteins (Bromodomain containing proteins BRD2 and BRD4) interact with modified chromatin. We are providing experimental chromatin biochemistry and Cell Biological tools and reagents that are used in Peter DiMaggio's lab to identify chromatin modifications that are recognised by BRD2 and BRD4 by mass spectrometric and computational approaches.
Collaborator Contribution GSK/Epinova have provided us with reagents to investigate the impact of Bromodomain Inhibitors on the interaction of BRD proteins with modified chromatin. Peter DiMaggio's lab provides expertise in mass spectrometry and computational approaches that allow the identification of chromatin modifications that are recognised by BRD2 and BRD4.
Impact This is a highly interdisciplinary project combining the expertise in small molecule applications of a large Pharmaceutical Company (GSK) with our expertise in Chromatin Biochemistry and Peter DiMaggio's expertise in mass spectrometry and computational methods. Our current output is the high level interdisciplinary training of a PhD student who is involved in a project combining Cell Biology, Biochemistry, Mass Spectrometry and computational approaches.
Start Year 2015
 
Description Collaboration with Peter DiMaggio/Chemical Engineering (Imperial College) 
Organisation Imperial College London (ICL)
Department Department of Chemical Engineering
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Co-supervision of two PhD students together with Peter DiMaggio (Dept. of Chemical Engineering) conducting research on a novel chemical cross-linking and mass spectrometry approach to elucidate the composition and architecture of multi-subunit chromatin regulator complexes. We contribute the experimental chromatin biochemistry knowledge and methods and purified protein complexes to be used in the mass spectrometric analyses.
Collaborator Contribution Co-supervision of two PhD students conducting research on a novel chemical cross-linking and mass spectrometry approach to elucidate the composition and architecture of multi-subunit chromatin regulator complexes. Peter DiMaggio's group at the Dept. of Chemical Engineering contributes experimental mass-spectrometry expertise and instrumentation and computational analysis of mass spectrometry data.
Impact We are co-supervising two jointly supervised PhD students conducting a research project to develop a novel chemical cross-linking and mass spectrometry approach to elucidate the composition and architecture of multi-subunit chromatin regulator complexes. The project is highly interdisciplinary and involves Molecular Biology, Chemical Biology, Chromatin Biology, Protein Biochemistry and Cell Biology approaches on our side and advanced Chemical Synthesis, Mass Spectrometry and Computation Approaches on Peter DiMaggio's side. The current output consists of high level interdisciplinary training of the two PhD students who are involved in a project combining biological, biochemical, mass-spectrometric and computational methods.
Start Year 2012
 
Description Collaboration with Richard Jenner, University College London 
Organisation University College London (UCL)
Department UCL Biochemical Engineering
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have provided reagents for nucleosome pulldowns for investigating the RNA-dependence of the interaction of PRC2 with chromatin
Collaborator Contribution Our partners carried out the nucleosome pulldown experiments
Impact This collaboration has resulted in the publication "Beltran M, Yates CM, Skalska L, Dawson M, Reis FP, Viiri K, Fisher CL, Sibley CR, Foster BM, Bartke T, Ule J, and Jenner RG. (2016). The interaction of PRC2 with RNA or chromatin is mutually antagonistic. Genome Res. 26, 896-907. DOI: 10.1101/gr.197632.115."
Start Year 2015
 
Description Epigenesys Associate Member 
Organisation European Commission (EC)
Department EpiGeneSys
Country European Union (EU) 
Sector Learned Society 
PI Contribution We do not make any formal contribution except for participation in network conferences
Collaborator Contribution Lab members can participate in network conferences and workshops (costs for the conference and accommodation are usually covered by the network)
Impact None
Start Year 2013