University of Bradford eThesis This thesis is hosted in Bradford Scholars – The University of Bradford Open Access repository. Visit the repository for full metadata or to contact the repository team © University of Bradford. This work is licenced for reuse under a Creative Commons Licence. AN INTEGRATIVE BIOINFORMATICS APPROACH FOR ANALYSES OF MULTI-LEVEL TRANSCRIPTIONAL REGULATION AND THREE-DIMENSIONAL GENOME ORGANIZATION IN THE EPIDERMIS AND SKIN APPENDAGES Exploring genomic transcriptional profiles of the distinct stages of hair follicle and sweat gland development and analyses of mechanism integrating the transcriptional regulation, linear and high-order genome organization within epidermal differentiation complex in keratinocytes Krzysztof POTERLOWICZ submitted for the degree of Doctor of Philosophy School of Life Sciences University of Bradford 2013 Abstract An integrative bioinformatics approach for analyses of multi-level transcriptional regulation and three-dimensional organization in the epidermis and skin appendages by Krzysztof Poterlowicz Keywords: Bioinformatics, microarray, ChIP-on-chip, chromosome conformation capture carbon copy (5C), transcriptional regulation, three- dimensional genome organization The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to i hair follicles, suggesting substantial differences in global genome re- arrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factor- mediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin. ii Acknowledgments First and most important I would like to thank you to my supervisor Professor Vladimir Botchkarev for his encouragement, cooperation and support for the last three years. I am particular grateful for his patience, while introducing a non-biologist into amazing field of skin genetics and epigenetics. I am especially grateful to my secondary supervisor Dr Yonghong Peng for bringing me into field of bioinformatics. I would like to thank Dr Michael Fessing for his scientific and academic advices. I would like to express my special thanks to my dearest friends Dr Michal Gdula and Dr Julian Laubenthal. I am very grateful to Dr Andrei Mardaryev, Dr Mohammed Ahmed, Dr Andrey Sharov, Dr Tanya Sharova for generation of experimental data used in this thesis and publications. I would like to show my appreciation to Dr Natasha Botchkareva for her help. I would like to show my gratitude to Professor Terumi Kohwi-Shigematsu (National Laurence Berkley Laboratory, University of California Berkley) for successful and productive collaboration. I would like to thank you my lovely wife Gosia for her tremendous support, love and extraordinary patience and also thanks for giving me "joy of life" to my children Paulina and Patryk. iii Finally, I would like to appreciate my parents and my brother for their support. iv Publications 1. M.R. Gdula, K. Poterlowicz, A.N. Mardaryev, A.A. Sharov, Y. Peng, M.Y. Fessing, V.A. Botchkarev “Remodeling of three-dimensional organization of the nucleus during terminal keratinocyte differentiation in the epidermis”, Journal of Investigative Dermatology, 2013 [Epub ahead of print] [IF: 6.270] 2. T. Kohwi-Shigematsu, K. Poterlowicz, E. Ordinario, H.J. Han, V.A. Botchkarev, Y. Kohwi “Genome organizing function of SATB1 in tumor progression.” Seminars in Cancer Biology, 2012 [Epub ahead of print] [IF: 6.475] 3. A.N. Mardaryev, N. Meier, K. Poterlowicz, A.A. Sharov, T. Sharova, M.I. Ahmed, V. Rapisarda, C. Lewis, M.Y. Fessing, T.M. Ruenger, J. Bhawan, S. Werner, R. Paus and V.A. Botchkarev “Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after Injury” Development, 2011 Nov; 138(22): 4843-52 [IF: 7.467] 4. M.Y. Fessing, A.N. Mardaryev, M.R. Gdula, A.A. Sharov, T Sharova, V. Rapisarda, K.B. Gordon, A.D Smorodchenko, K. Poterlowicz, G. Ferone, Y. Kohwi, C. Missero, T. Kohwi-Shigematsu and V.A. Botchkarev “p63 Regulates Satb1 to control Tissue-Specific Chromatin Remodeling during Development of the Epidermis”, Journal of Cell Biology, 2011 Sep; 194(6): 825-39. [IF: 10.123] v 5. M.Y. Fessing, R. Atoyan, B. Shander, A.N. Mardaryev, V.V. Jr Botchkarev, K. Poterlowicz, Y. Peng, T. Efimova and V.A. Botchkarev “Bmp signaling induces cell-type specific changes in gene expression programs of human keratinocytes and fibroblasts”, Journal of Investigative Dermatology, 2010 Feb; 130(2): 398-404. [IF: 6.270] vi Abbreviations 3C Chromosome Conformation Capture 4C Circularized Chromosome Conformation Capture 5C Chromosome Conformation Capture Carbon Copy ChIP –on-chip Chromatin Immunoprecipitation Chip ChIP-seq Chromatin Immunoprecipitation Sequencing DNA Deoxyribonucleic acid EDC Epidermal Differentiation Complex FDR False Discovery Rate FP Foot Pad HFP Hair Follicle Placode HFM Hair Follicle Matrix mRNA Messenger RNA MC Monte Carlo RNA Ribonucleic acid PCR Polymerase chain reaction SGP Sweat Gland Placode SGA Sweat Gland Adult vii Table of Contents Abstract ............................................................................................................ i Acknowledgments ........................................................................................ iii Publications .................................................................................................... v Abbreviations ............................................................................................... vii Table of Contents ........................................................................................ viii List of Tables .............................................................................................. xiiii List of Figures .............................................................................................. xv 1 Introduction ............................................................................................... 1 1.1 Background ........................................................................................ 1 1.2 Aims and objectives .......................................................................... 2 1.3 Structure of thesis ............................................................................. 3 2 Literature review ....................................................................................... 4 2.1 Molecular mechanisms that control development of the skin and epidermal appendages (hair follicles and sweat glands) ....................... 5 2.1.1 Molecular mechanisms that control development of the epidermis 7 2.1.2 Signaling, transcription factor-mediated and epigenetic regulatory mechanisms that control epidermal development .................................. 12 2.1.3 Mechanisms controlling the development of epidermal appendages (hair follicles and sweat glands) ......................................... 18 viii 2.2 Integration of the transcription factor-mediated and epigenetic regulatory mechanisms that control keratinocyte differentiation in the epidermis and hair follicles ..................................................................... 33 2.2.1 DNA methylation .......................................................................... 39 2.2.2 Covalent histone modifications ..................................................... 41 2.2.3 Three-dimensional genome organization and higher-order chromatin remodelling ............................................................................ 43 2.2.4 Links between the epigenetic and transcription factor-mediated regulatory mechanisms in the control of keratinocyte differentiation ...... 45 2.3 Methods and approaches used for analyses of the global microarray, ChIP-on-chip and 4C-seq data in mouse cutaneous biology research ....................................................................................... 47 3 Material and Methods ............................................................................. 52 3.1 Generation of the