DOCSLIB.ORG

Promiscuous Gene Expression in the Thymic Medulla – on Regulation at the Epigenetic and Single Cell Level

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Promiscuous Gene Expression in the Thymic Medulla – on Regulation at the Epigenetic and Single Cell Level Promiscuous gene expression in the thymic medulla – on regulation at the epigenetic and single cell level INAUGURAL – DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich - Mathematischen Gesamtfakultät der Ruprecht – Karls – Universität Heidelberg vorgelegt von Diplom-Biochemikerin Anna Sinemus aus Göttingen Gutachter: Prof. Dr. Günter Hämmerling Prof. Dr. Bruno Kyewski Die vorliegende Arbeit wurde angefertigt in der Abteilung Entwicklungsimmunologie, Leitung Prof. Dr. Bruno Kyewski, im Deutschen Krebsforschungszentrum Heidelberg. Hiermit erkläre ich, dass ich die vorgelegte Dissertation selbst verfasst und mich dabei keiner anderen, als der von mir ausdrücklich bezeichneten Quellen bedient habe. Heidelberg, Anna Sinemus _______________________________________________________________________CONTENTS Contents ZUSAMMENFASSUNG ............................................................................................. 5 SUMMARY................................................................................................................. 6 LIST OF ABBREVIATIONS ....................................................................................... 7 1. INTRODUCTION .................................................................................................... 9 1.1 Thymocyte maturation and central T cell tolerance.................................................................................... 9 1.1.1 T cell maturation and selection.................................................................................................................. 9 1.1.2 Dominant Tolerance................................................................................................................................ 12 1.2 Promiscuous gene expression ....................................................................................................................... 12 1.2.1 Regulation of pGE at the cellular level ................................................................................................... 13 1.2.2 Regulation of pGE at the molecular level ............................................................................................... 15 1.3 Mechanisms of epigenetic regulation........................................................................................................... 18 1.3.1 DNA methylation .................................................................................................................................... 18 1.3.2 The histone code...................................................................................................................................... 19 1.3.3 The role of chromatin structure and nuclear organization....................................................................... 20 1.4 Objective of this study .................................................................................................................................. 22 2. MATERIALS AND METHODS ............................................................................. 24 2.1 Materials ........................................................................................................................................................ 24 2.1.1 Chemicals................................................................................................................................................ 24 2.1.2 Buffers and commercial solutions........................................................................................................... 25 2.1.3 Enzymes, Proteins ................................................................................................................................... 26 2.1.3 Antibodies, dyes...................................................................................................................................... 27 2.1.4 Primers and (Oligo-) Nucleotides............................................................................................................ 27 2.1.5 Commercial Kits ..................................................................................................................................... 30 2.1.6 Mice, cell lines, bacteria.......................................................................................................................... 30 2.1.7 Consumables ........................................................................................................................................... 31 2.1.8 Equipment ............................................................................................................................................... 31 2.1.9 Software .................................................................................................................................................. 32 2.2 Methods.......................................................................................................................................................... 33 2.2.1 Distance measurements using fluorescence – in situ – hybridization (FISH) ......................................... 33 2.2.2. Agarose gel electrophoresis.................................................................................................................... 39 2.2.3 RNA isolation.......................................................................................................................................... 40 2.2.4 RT-PCR................................................................................................................................................... 40 2.2.5 Isolation of genomic DNA from murine tails.......................................................................................... 40 2.2.6 “Conventional” and Quantitatve PCR (qPCR)........................................................................................ 41 2.2.7 Single-cell PCR (SC PCR) ...................................................................................................................... 42 2.2.8 Chromatin-Immunoprecipitation............................................................................................................. 45 _______________________________________________________________________CONTENTS 2.2.9 Counting of live cells .............................................................................................................................. 47 2.2.10 Antibody labeling.................................................................................................................................. 47 2.2.11 Isolation of specific cell populations from mice.................................................................................... 47 2.2.12 Immunohistochemistry.......................................................................................................................... 50 3. RESULTS............................................................................................................. 52 3.1 Chromatin structure is modified at the level of individual genes, not the whole locus ........................... 52 3.1.1 The casein gene locus as a model locus for promiscuous gene expression ............................................. 52 3.1.2 Optimization of the ChIP protocol for low numbers of ex vivo sorted cells............................................ 53 3.1.3 Active chromatin marks are present only at the Casein beta promoter ................................................... 54 3.1.4 Detection threshold of ChIP is above the frequency of most promiscuously expressed genes in the casein locus ................................................................................................................................................................. 57 3.1.5 Enrichment of mTEC expressing a single antigen: Chromatin marks in Gad67/eGFP mice .................. 58 3.2 Expression patterns of a single promiscuously expressed antigen – lessons from the Gad67 locus using Gad67-eGFP knock-in mice ............................................................................................................................... 61 3.2.1 Co-enrichment for Gad67 expression in eGFP+ mTEChigh on the population level................................. 61 3.2.2 SC PCR analysis of the Gad67 locus shows decoupling between eGFP protein and mRNA expression 62 3.2.3 Strong preference for biallelic expression at the Gad67 locus ................................................................ 63 3.2.4 Gad67/eGFP co-expressors are enriched in Aire+ cells........................................................................... 65 3.2.5 Gad67 and eGPF are co-expressed at the protein level in the brain ........................................................ 65 3.3 Nuclear positioning and DNA compaction of the casein locus during mTEC maturation..................... 67 3.3.1 Two probes are localized upstream and downstream of Csna/Csnb to measure chromatin decompaction .......................................................................................................................................................................... 68 3.3.2 Both decompaction of the locus and nuclear localization can be measured with the same probes ......... 68 3.3.3 Chromatic shifts between Oregon Green-Alexa Fluor 647 are small when directly measured in parallel with probes....................................................................................................................................................... 71 3.3.4 Chromatin in the Casein locus is more compact
Load more

Recommended publications
  • Modeling of Histone Modifications Reveals Formation Mechanism and Function of Bivalent Chromatin
    bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429504; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Modeling of histone modifications reveals formation mechanism and function of bivalent chromatin Wei Zhao1,2, Lingxia Qiao1, Shiyu Yan2, Qing Nie3,*, Lei Zhang1,2,* 1 Beijing International Center for Mathematical Research, Peking University, Beijing 100871, China 2 Center for Quantitative Biology, Peking University, Beijing 100871, China 3 Department of Mathematics and Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697, USA. *Corresponding authors: Qing Nie, email: [email protected]; Lei Zhang, email: [email protected] Abstract Bivalent chromatin is characterized by occupation of both activating histone modifications and repressive histone modifications. While bivalent chromatin is known to link with many biological processes, the mechanisms responsible for its multiple functions remain unclear. Here, we develop a mathematical model that involves antagonistic histone modifications H3K4me3 and H3K27me3 to capture the key features of bivalent chromatin. Three necessary conditions for the emergence of bivalent chromatin are identified, including advantageous methylating activity over demethylating activity, frequent noise conversions of modifications, and sufficient nonlinearity. The first condition is further confirmed by analyzing the
    [Show full text]
  • Epigenetic Regulation of Promiscuous Gene Expression in Thymic Medullary Epithelial Cells
    Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells Lars-Oliver Tykocinskia,1,2, Anna Sinemusa,1, Esmail Rezavandya, Yanina Weilandb, David Baddeleyb, Christoph Cremerb, Stephan Sonntagc, Klaus Willeckec, Jens Derbinskia, and Bruno Kyewskia,3 aDivision of Developmental Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany; bKirchhoff Institute for Physics, University of Heidelberg, D-69120 Heidelberg, Germany; and cInstitute for Genetics, University of Bonn, D-53117 Bonn, Germany Edited* by Philippa Marrack, National Jewish Health, Denver, CO, and approved September 28, 2010 (received for review July 2, 2010) Thymic central tolerance comprehensively imprints the T-cell re- ing of delimited regions allowing access of general and specific ceptor repertoire before T cells seed the periphery. Medullary transcriptional factors to act on gene-specific control elements thymic epithelial cells (mTECs) play a pivotal role in this process by (8). This scenario is clearly different from the intricate regulation virtue of promiscuous expression of tissue-restricted autoantigens. of functionally related gene families like the Hox gene locus or β The molecular regulation of this unusual gene expression, in the -globin gene locus (9). A similar phenomenon as observed in Drosophila particular the involvement of epigenetic mechanisms is only poorly has been reported for housekeeping genes but not for understood. By studying promiscuous expression of the mouse TRAs in vertebrates (10). casein locus, we report that transcription of this locus proceeds Here we analyzed the interrelationship between emerging gene expression patterns at the single cell level, promoter-associated from a delimited region (“entry site”) to increasingly complex pat- epigenetic marks, and the differentiation of mTECs in the murine terns along with mTEC maturation.
    [Show full text]
  • Histone H3k27me3 Demethylases Regulate Human Th17 Cell Development and Effector Functions by Impacting on Metabolism
    Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism Adam P. Cribbsa,1, Stefan Terlecki-Zaniewicza, Martin Philpotta, Jeroen Baardmanb, David Ahernc, Morten Lindowd, Susanna Obadd, Henrik Oerumd, Brante Sampeye, Palwinder K. Manderf, Henry Penng, Paul Wordswortha, Paul Bownessa, Menno de Wintherh, Rab K. Prinjhaf, Marc Feldmanna,c,1, and Udo Oppermanna,i,j,k,1 aBotnar Research Center, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, National Institute for Health Research Oxford Biomedical Research Unit, University of Oxford, OX3 7LD Oxford, United Kingdom; bExperimental Vascular Biology, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centres, University of Amsterdam, 1105AZ Amsterdam, The Netherlands; cKennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, National Institute for Health Research Oxford Biomedical Research Unit, University of Oxford, OX3 7FY Oxford, United Kingdom; dRoche Innovation Center Copenhagen A/S, DK 2970 Hørsholm, Denmark; eMetabolon Inc., Durham, NC 27713; fEpinova Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, SG1 2NY Stevenage, United Kingdom; gArthritis Centre, Northwick Park Hospital, HA13UJ Harrow, United Kingdom; hInstitute for Cardiovascular Prevention, Ludwig Maximilians University, 80336 Munich, Germany; iStructural Genomics Consortium, University of Oxford, OX3 7DQ Oxford,
    [Show full text]
  • Dual Recognition of H3k4me3 and H3k27me3 by a Plant Histone Reader SHL
    ARTICLE DOI: 10.1038/s41467-018-04836-y OPEN Dual recognition of H3K4me3 and H3K27me3 by a plant histone reader SHL Shuiming Qian1,2, Xinchen Lv3,4, Ray N. Scheid1,2,LiLu1,2, Zhenlin Yang3,4, Wei Chen3, Rui Liu3, Melissa D. Boersma2, John M. Denu2,5,6, Xuehua Zhong 1,2 & Jiamu Du 3 The ability of a cell to dynamically switch its chromatin between different functional states constitutes a key mechanism regulating gene expression. Histone mark “readers” display 1234567890():,; distinct binding specificity to different histone modifications and play critical roles in reg- ulating chromatin states. Here, we show a plant-specific histone reader SHORT LIFE (SHL) capable of recognizing both H3K27me3 and H3K4me3 via its bromo-adjacent homology (BAH) and plant homeodomain (PHD) domains, respectively. Detailed biochemical and structural studies suggest a binding mechanism that is mutually exclusive for either H3K4me3 or H3K27me3. Furthermore, we show a genome-wide co-localization of SHL with H3K27me3 and H3K4me3, and that BAH-H3K27me3 and PHD-H3K4me3 interactions are important for SHL-mediated floral repression. Together, our study establishes BAH-PHD cassette as a dual histone methyl-lysine binding module that is distinct from others in recognizing both active and repressive histone marks. 1 Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA. 2 Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53706, USA. 3 National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China.
    [Show full text]
  • The Importance of Epigenetics in Diagnostics and Treatment of Major Depressive Disorder
    Journal of Personalized Medicine Review The Importance of Epigenetics in Diagnostics and Treatment of Major Depressive Disorder Piotr Czarny 1,† , Katarzyna Białek 2,†, Sylwia Ziółkowska 1,†, Justyna Strycharz 1 , Gabriela Barszczewska 2 and Tomasz Sliwinski 2,* 1 Department of Medical Biochemistry, Medical University of Lodz, 92-216 Lodz, Poland; [email protected] (P.C.); [email protected] (S.Z.); [email protected] (J.S.) 2 Laboratory of Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz, 90-236 Lodz, Poland; [email protected] (K.B.); [email protected] (G.B.) * Correspondence: [email protected] † Equal contribution. Abstract: Recent studies imply that there is a tight association between epigenetics and a molecular mechanism of major depressive disorder (MDD). Epigenetic modifications, i.e., DNA methylation, post-translational histone modification and interference of microRNA (miRNA) or long non-coding RNA (lncRNA), are able to influence the severity of the disease and the outcome of the therapy. This article summarizes the most recent literature data on this topic, i.e., usage of histone deacetylases as therapeutic agents with an antidepressant effect and miRNAs or lncRNAs as markers of depression. Due to the noteworthy potential of the role of epigenetics in MDD diagnostics and therapy, we have gathered the most relevant data in this area. Citation: Czarny, P.; Białek, K.; Ziółkowska, S.; Strycharz, J.; Keywords: depression; epigenetics; personalized medicine; histones; histone modifications; DNA Barszczewska, G.; Sliwinski, T. The methylation; microRNA; long non-coding RNA Importance of Epigenetics in Diagnostics and Treatment of Major Depressive Disorder.
    [Show full text]
  • Dynamics of Transcription-Dependent H3k36me3 Marking by the SETD2:IWS1:SPT6 Ternary Complex
    bioRxiv preprint doi: https://doi.org/10.1101/636084; this version posted May 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dynamics of transcription-dependent H3K36me3 marking by the SETD2:IWS1:SPT6 ternary complex Katerina Cermakova1, Eric A. Smith1, Vaclav Veverka2, H. Courtney Hodges1,3,4,* 1 Department of Molecular & Cellular Biology, Center for Precision Environmental Health, and Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, 77030, USA 2 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic 3 Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA 4 Department of Bioengineering, Rice University, Houston, TX, 77005, USA * Lead contact; Correspondence to: [email protected] Abstract The genome-wide distribution of H3K36me3 is maintained SETD2 contributes to gene expression by marking gene through various mechanisms. In human cells, H3K36 is bodies with H3K36me3, which is thought to assist in the mono- and di-methylated by eight distinct histone concentration of transcription machinery at the small portion methyltransferases; however, the predominant writer of the of the coding genome. Despite extensive genome-wide data trimethyl mark on H3K36 is SETD21,11,12. Interestingly, revealing the precise localization of H3K36me3 over gene SETD2 is a major tumor suppressor in clear cell renal cell bodies, the physical basis for the accumulation, carcinoma13, breast cancer14, bladder cancer15, and acute maintenance, and sharp borders of H3K36me3 over these lymphoblastic leukemias16–18. In these settings, mutations sites remains rudimentary.
    [Show full text]
  • Early Life Stress Induces Age-Dependent Epigenetic Changes
    www.nature.com/scientificreports OPEN Early life stress induces age‑dependent epigenetic changes in p11 gene expression in male mice Mi Kyoung Seo1, Jung Goo Lee1,2* & Sung Woo Park1,3* Early life stress (ELS) causes long‑lasting changes in gene expression through epigenetic mechanisms. However, little is known about the efects of ELS in adulthood, specifcally across diferent age groups. In this study, the epigenetic modifcations of p11 expression in adult mice subjected to ELS were investigated in diferent stages of adulthood. Pups experienced maternal separation (MS) for 3 h daily from postnatal day 1 to 21. At young and middle adulthood, behavioral test, hippocampal p11 expression levels, and levels of histone acetylation and methylation and DNA methylation at the hippocampal p11 promoter were measured. Middle‑aged, but not young adult, MS mice exhibited increased immobility time in the forced swimming test. Concurrent with reduced hippocampal p11 levels, mice in both age groups showed a decrease in histone acetylation (AcH3) and permissive histone methylation (H3K4me3) at the p11 promoter, as well as an increase in repressive histone methylation (H3K27me3). Moreover, our results showed that the expression, AcH3 and H3Kme3 levels of p11 gene in response to MS were reduced with age. DNA methylation analysis of the p11 promoter revealed increased CpG methylation in middle‑aged MS mice only. The results highlight the age‑ dependent deleterious efects of ELS on the epigenetic modifcations of p11 transcription. Children exposed to early life stress (ELS) such as neglect and abuse have a signifcantly increased risk of devel- oping depression1,2. In human and animal studies, ELS has been reported to induce a depression-like phenotype in adulthood3,4.
    [Show full text]
  • Deciphering the Histone Code to Build the Genome Structure
    bioRxiv preprint doi: https://doi.org/10.1101/217190; this version posted November 13, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Deciphering the histone code to build the genome structure Kirti Prakasha,b,c,* and David Fournierd,* aPhysico-Chimie Curie, Institut Curie, CNRS UMR 168, 75005 Paris, France; bOxford Nanoimaging Ltd, OX1 1JD Oxford, UK; cMicron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, Oxford, UK; dFaculty of Biology and Center for Computational Sciences, Johannes Gutenberg University Mainz, 55128 Mainz, Germany; *Correspondence: [email protected], [email protected] Histones are punctuated with small chemical modifications that alter their interaction with DNA. One attractive hypothesis stipulates that certain combinations of these histone modifications may function, alone or together, as a part of a predictive histone code to provide ground rules for chromatin folding. We consider four features that relate histone modifications to chromatin folding: charge neutrali- sation, molecular specificity, robustness and evolvability. Next, we present evidence for the association among different histone modi- fications at various levels of chromatin organisation and show how these relationships relate to function such as transcription, replica- tion and cell division. Finally, we propose a model where the histone code can set critical checkpoints for chromatin to fold reversibly be- tween different orders of the organisation in response to a biological stimulus. DNA | nucleosomes | histone modifications | chromatin domains | chro- mosomes | histone code | chromatin folding | genome structure Introduction The genetic information within chromosomes of eukaryotes is packaged into chromatin, a long and folded polymer of double-stranded DNA, histones and other structural and non- structural proteins.
    [Show full text]
  • Nucleosome Eviction Along with H3k9ac Deposition Enhances Sox2 Binding During Human Neuroectodermal Commitment
    Cell Death and Differentiation (2017) 24, 1121–1131 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cdd Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment Yanhua Du1,2, Zhenping Liu2, Xinkai Cao2, Xiaolong Chen2, Zhenyu Chen3, Xiaobai Zhang2, Xiaoqing Zhang*,1,3 and Cizhong Jiang*,1,2 Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling. Cell Death and Differentiation (2017) 24, 1121–1131; doi:10.1038/cdd.2017.62; published online 5 May 2017 Chromatin remodeling offers the epigenetic basis for tran- that H3K4me3 and H3K27me3 effectively distinguish genes scriptional regulation and has a pivotal role in many biological with different expression levels and reflect lineage potential.11 A processes such as cell differentiation, embryonic develop- recent HPLC-MS-based quantitative proteomics identified ment, and so on.
    [Show full text]
  • New Insights Into the Role of Histone Changes in Aging
    International Journal of Molecular Sciences Review New Insights into the Role of Histone Changes in Aging Sun-Ju Yi and Kyunghwan Kim * Department of Biology, School of Biological Sciences, College of Natural Sciences, Chungbuk National University, Cheongju 28644, Chungbuk, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-(43)-2612292 Received: 14 October 2020; Accepted: 2 November 2020; Published: 3 November 2020 Abstract: Aging is the progressive decline or loss of function at the cellular, tissue, and organismal levels that ultimately leads to death. A number of external and internal factors, including diet, exercise, metabolic dysfunction, genome instability, and epigenetic imbalance, affect the lifespan of an organism. These aging factors regulate transcriptome changes related to the aging process through chromatin remodeling. Many epigenetic regulators, such as histone modification, histone variants, and ATP-dependent chromatin remodeling factors, play roles in chromatin reorganization. The key to understanding the role of gene regulatory networks in aging lies in characterizing the epigenetic regulators responsible for reorganizing and potentiating particular chromatin structures. This review covers epigenetic studies on aging, discusses the impact of epigenetic modifications on gene expression, and provides future directions in this area. Keywords: aging; histone level; histone modification; histone variant; chromatin remodeling; epigenetics 1. Introduction An individual organism undergoes a series of developmental stages, including birth, growth, maturity, aging, and death. Aging is the gradual and continuous decline or loss of function at the cellular, tissue, and organismal levels with the passage of time. Aging is considered to begin in early adulthood, but is regarded as an integral part of life since other stages also affect the aging process [1].
    [Show full text]
  • Epigenomic Analysis Reveals DNA Motifs Regulating Histone Modifications in Human and Mouse
    Epigenomic analysis reveals DNA motifs regulating histone modifications in human and mouse Vu Ngoa,1, Zhao Chenb,1, Kai Zhanga, John W. Whitakerb, Mengchi Wanga, and Wei Wanga,b,c,2 aGraduate Program of Bioinformatics and Systems Biology, University of California, San Diego, La Jolla, CA 92093-0359; bDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0359; and cDepartment of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0359 Edited by Steven Henikoff, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved January 3, 2019 (received for review August 6, 2018) Histones are modified by enzymes that act in a locus, cell-type, and An analogy is that a transcription factor (TF) recognizes the same developmental stage-specific manner. The recruitment of enzymes DNA motif but its binding sites are cell-type–dependent. However, if to chromatin is regulated at multiple levels, including interaction we identify all motifs enriched in the TF binding sites across a large with sequence-specific DNA-binding factors. However, the DNA- and diverse set of cell types, the most common motif is likely the one binding specificity of the regulatory factors that orchestrate spe- recognized by the TF. Histone modifications are more complicated cific histone modifications has not been broadly mapped. We have than a single TF binding and one histone mark can be regulated by analyzed 6 histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, K3H9me3, H3K36me3) across 121 human cell types and tissues from multiple factors recognizing different motifs. Therefore, a compar- the NIH Roadmap Epigenomics Project as well as 8 histone marks ative analysis across diverse cell types/tissues is critical.
    [Show full text]
  • Downloaded from Were Then Stained with Primary Antibodies (2 H, Room Temperature), Washed and GSE9259765
    ARTICLE https://doi.org/10.1038/s41467-021-24158-w OPEN Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2 Thomas Barthlott1,8, Adam E. Handel 2,8, Hong Ying Teh1,8, Rushika C. Wirasinha3, Katrin Hafen1, Saulius Žuklys1, Benoit Roch4, Stuart H. Orkin 5, Jean-Pierre de Villartay 4, Stephen R. Daley3,7 & ✉ Georg A. Holländer 1,6 1234567890():,; Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold. 1 Department of Biomedicine and University Children’s Hospital of Basel, University of Basel, Basel, Switzerland. 2 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK. 3 Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia.
    [Show full text]