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Open Dissertation Tejaswinimishra.Pdf The Pennsylvania State University The Graduate School College of Science INTEGRATIVE GENOME-WIDE STUDIES TO ELUCIDATE REGULATION OF LINEAGE CHOICE IN HEMATOPOIESIS A Dissertation in Cell and Developmental Biology by Tejaswini Mishra ©2014 Tejaswini Mishra Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2014 ii The dissertation of Tejaswini Mishra was reviewed and approved* by the following: Ross Hardison T. Ming Chu Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Michael Axtell Associate Professor of Biology Debashis Ghosh Professor of Statistics, Public Health Sciences Robert F. Paulson Professor of Veterinary and Biomedical Sciences B. Franklin Pugh Willaman Chair in Molcular Biology Professor of Biochemistry and Molecular Biology Zhi-Chun Lai Professor of Biology, Biochemistry and Molecular Biology. Chair, Intercollege Graduate Degree Program in Cell and Developmental Biology *Signatures are on file in the Graduate School iii ABSTRACT Regulation of gene expression in multicellular eukaryotes allows their cells to express heterogeneous transcriptomes despite possessing the same genome, thus resulting in tissue-specific gene expression, which in turn drives cellular differentiation. Hematopoiesis in mouse is an ideal system in which to study specification of cell fate and cellular differentiation after lineage commitment. Differential gene expression drives lineage commitment and maturation during differentiation, but few studies have addressed changes in gene expression genome-wide across these processes. Much is known about the transcriptome landscape of differentiated erythroblasts and megakaryocytes; however, the transcriptome of the megakaryocyte-erythroid progenitor is relatively unexplored. Till date, comparative transcriptome studies elucidating the alteration in transcriptional output between bipotential progenitors and differentiated, monopotent erythroblasts and megakaryocytes have not been performed. Additionally, even though these sister lineages are regulated by a common set of well-studied transcription factors, it is still unclear as to how these factors exert lineage-specific actions. I have examined changes in the transcriptome during the commitment of the bipotential megakaryocyte-erythroid progenitor into its daughter lineages to infer models of how these changes drive commitment to either of two radically distinct lineage outcomes. I used RNA-seq to map the transcriptome of the bipotential megakaryocyte-erythroid progenitor (MEP) prior to commitment to its two daughter lineages and also the transcriptomes of maturing erythroblasts (ERY) and megakaryocytes (MEG) after commitment. Comparison of these transcriptome maps revealed that MEPs already express much of the MEG program while continuing to express genes associated parallel myeloid lineages such as granulocytes. In contrast, greater numbers of genes are induced in ERY than MEG, along with repression of pan-hematopoietic genes and genes involved in proliferation, signaling, and cell growth. These results suggest a model of broad expression of genes iv in MEPs that are both a memory of previous myeloid potential and permissive for MEG differentiation, while active induction and repression are needed to execute the erythroid program. This model is supported by genome-wide maps of transcription factor (TF) occupancy. Genes specifically expressed in MEG were preferentially occupied by TFs in early, multipotent hematopoietic progenitors and continue to maintain occupancy post-commitment, whereas erythroid genes were primarily occupied in committed erythroid cells. These results suggest that the default commitment outcome for MEP is MEG, and commitment to ERY requires a radical rewiring of transcription circuitry. v TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………………………..vii LIST OF TABLES…………………………………………………………………………...xii ACKNOWLEDGEMENTS………………………………………………………………....xiii Chapter 1 Introduction ........................................................................................................... 1 1.1 Hematopoiesis as a model to study lineage commitment and differentiation ......... 4 1.2 General biology of erythroid and megakaryocytic cells .......................................... 5 1.3 Lineage commitment in hematopoietic systems: regulatory paradigms and common themes .................................................................................................................... 11 1.3.1 Models of commitment in hematopoiesis ..................................................... 11 1.3.2 Lineage priming ............................................................................................ 13 1.3.3 Lineage plasticity .......................................................................................... 14 1.3.4 Role of cytokines .......................................................................................... 15 1.3.5 Actions of TFs during lineage commitment ................................................. 16 1.3.6 Lineage commitment in a nutshell ................................................................ 17 1.4 Control of transcription during lineage commitment: ............................................. 18 1.4.1 Typical transcription patterns during lineage commitment .......................... 18 1.4.2 Transcription factors regulating hematopoiesis ............................................ 18 1.4.2.1 GATA1 .............................................................................................. 19 1.4.2.2 FOG1 .................................................................................................. 22 1.4.2.3 TAL1 .................................................................................................. 23 1.4.2.4 FLI1 .................................................................................................... 24 1.4.2.5 EKLF .................................................................................................. 24 1.4.2.6 GATA2 .............................................................................................. 25 1.4.2.7 ETO2 .................................................................................................. 25 1.4.2.8 GFI1B ................................................................................................ 26 1.4.2.9 PU.1 ................................................................................................... 26 1.4.2.10 Other transcription factors ............................................................... 27 1.4.3 Chromatin accessibility ................................................................................. 28 1.4.4 Noncoding RNAs .......................................................................................... 29 1.4.5 Summary of gene regulation in hematopoiesis ............................................. 31 Chapter 2 Transcriptome profiling using RNA-seq: history, challenges and solutions ........ 35 2.1 Microarray-based assays of gene expression ........................................................... 37 2.2 RNA-seq as a tool to study transcriptomes: advantages, and applications .............. 39 2.3 Typical RNA-seq data processing pipeline for Illumina sequencing ...................... 45 2.3.1 Quality assessment ........................................................................................ 46 2.3.2 Mapping ........................................................................................................ 46 2.3.3 Transcript assembly ...................................................................................... 48 2.3.4 Quantification of expression levels ............................................................... 49 2.3.5 Differential expression testing ...................................................................... 50 2.4 Scope of the present study: standardization of transcriptome analysis ................... 51 2.5 Hematopoietic cells assayed using RNA-seq .......................................................... 52 vi 2.5.1 Lineage commitment during erythromegakaryopoiesis ............................... 53 2.5.2 GATA1-dependent erythropoiesis and the G1E model for erythroid differentiation ................................................................................................. 53 2.5.3 Models of stress erythropoiesis ..................................................................... 55 2.6 Materials and Methods ............................................................................................ 55 2.6.1 Cell Culture ................................................................................................... 55 2.6.2 Primary cells isolation .................................................................................. 55 2.6.3 mRNA extraction and cDNA synthesis ........................................................ 56 2.6.4 Library Preparation and sequencing ............................................................. 57 2.6.5 Mapping ........................................................................................................ 57 2.6.6 Transcript assembly ...................................................................................... 58 2.6.7 ChIP-seq ........................................................................................................ 58 2.6.8 ChIP-seq peak calling ................................................................................... 59 2.6.9 Functional enrichments
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