Dissecting the epigenetic landscapes of hematopoiesis and fission yeast.

The genome of eukaryotic cells is stored in the nucleus as chromatin, a DNA-protein complex that serves to compact and protect the DNA molecules. The basic unit of chromatin is the nucleosome composed of DNA wrapped around a histone protein core. In addition to condensing and protecting the genome, chromatin confers a number of regulatory properties employed for example in control of gene expression and stabilization of repetitive sequences. Chromatin also constitutes an obstacle that needs to be negotiated in processes such as transcription elongation, DNA replication and DNA repair. A wide range of chromatin modifying factors and mechanisms are involved in regulating the state of chromatin and affect all DNA related processes. These mechanisms, often referred to as epigenetic, include methylation of DNA, regulation by non-coding RNAs, remodeling of nucleosomes, posttranslational modifications of histones and incorporation of variant histones. The resulting chromatin state is called the epigenome and can, in contrast to the underlying DNA sequence, differ between cells in the same organism. This thesis describes characterization of aspects of the egipenomes of hematopoietic cells and fission yeast. We show that in fission yeast, genes with related functions share common patterns of histone modifications in the promoter regions. We also demonstrate crosstalk between different histone modifications, including interdependence of histone H4 acetylation sites and regulatory roles of histone methylation for histone acetylation. To better understand how chromatin factors influence human blood development we analysed expression of genes encoding chromatin modifying proteins in the hematopoietic system, including the hematopoietic stem cells and a wide range of mature blood cells. In doing so we could identify epigenetic factors that were expressed in cell type, cell lineage or cancer specific patterns, implicating them in regulation of blood development. We also found that several genes display differential use of alternative transcription start sites between cell types. Finally we constructed an in-depth map of how DNA methylation and gene expression changes during human granulocyte development. Our experiments show that DNA methylation changes are linked to points of lineage restriction, implicating DNA methylation in control of cell fate. DNA methylation changes, most of which were decreases, were primarily located outside of CpG islands, which have been the focus of most DNA methylation studies historically. Interestingly, DNA methylation was especially dynamic in enhancer elements, and sites with decreasing DNA methylation overlapped with differentiation induced enhancers and increased expression of target genes. This result suggests a role of DNA methylation in regulating enhancer activity in granulopoiesis

Expression profile of G9A and p300 in leukemia and normal blood sample

DNA is allied with histone proteins to form nucleosomes and higher order structures available in eukaryotes. Histones having amino termini can be modified by acetylation. Acetylation of explicit residues of the histones is associated with gene activity and may play a elementary role in transcriptional regulation. Bromodomains, motifs found in several eukaryotic transcription factors, exclusively interact with acetyl-lysines in histones H3 and H4. p300 is functionally conserved transcriptional coactivators for various transcription factors and have intrinsic acetyltransferase activity. The covalent alteration of histone tails has regulatory roles in various nuclear processes, such as organization of transcription and mitotic chromosome condensation. Among the different groups of enzymes identified to catalyze the covalent modification, the most topical additions are the histone methyltransferases (HMTases), whose functions are now being characterized. G9A is a novel mammalian HMTase that prefer lysine. Specific chromosome translocations commonly found in human leukemia engross rearrangements of genes which are implicated in the regulation of hematopoiesis. Consequently, the chromosome translocations often results in the expression of gene products

DNA methyltransferases and their roles in tumorigenesis

Abstract DNA methylation plays an important role in gene expression, chromatin stability, and genetic imprinting. In mammals, DNA methylation patterns are written and regulated by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A and DNMT3B. Recent emerging evidence shows that defects in DNMTs are involved in tumor transformation and progression, thus indicating that epigenetic disruptions caused by DNMT abnormalities are associated with tumorigenesis. Herein, we review the latest findings related to DNMT alterations in cancer cells and discuss the contributions of these effects to oncogenic phenotypes

Doctor of Philosophy

dissertationProper cell fate decisions require precise and coordinated changes in gene expression. Alterations in gene expression proceed through the functions of transcription factors, their associated coregulators and histone modifying enzymes. However, how these complex and diverse groups of proteins and enzymes coordinate their respective functions at target genes remains largely unknown. To gain additional insights into the coordinated activities of transcription factors and histone modifying enzymes, we studied how the transcription factor growth factor independence 1 (GFI1) carries out transcriptional repression through interaction with coregulator histone modifying enzymes. GFI1 is a transcriptional repressor and master regulator of normal and malignant hematopoiesis. GFI1 is comprised of a transcriptionally repressive N-terminal Snail/Slug/GFI1 (SNAG) domain, a C-terminal concatemer of DNA binding zinc fingers and a linker region which separates them. The relatively simple protein domain structure of GFI1 makes it an ideal transcription factor for studying mechanisms of transcriptional repression. We describe here two novel mechanisms of transcriptional repression by GFI1, both of which occur through posttranslational modification. First, we identify and characterize a SUMOylation event carried out by the SUMO2/3, UBC9, and PIAS3 SUMOylation machinery. SUMOylation occurs at K239 within a type I SUMO consensus element in the linker region of GFI1. We find that SUMO iv defective GFI1 derivatives fail to complement Gfi1 depletion phenotypes in zebrafish developmental erythropoiesis and in granulocyte differentiation in cultured human cells. SUMO defective GFI1 derivatives also display impaired LSD1/CoREST binding and fail to repress the GFI1 target gene MYC during granulocyte differentiation and enforced MYC expression blocks GFI1 mediated granulocyte differentiation. Second, we show SMYD2 mediated methylation at K8 within the GFI1 SNAG domain is a critical determinant of GFI1 transcriptional repression and contributes to GFI1 hematopoietic differentiation and leukemia cell survival functions. The methylation defective GFI1 SNAG domain lacks repressor function due to a failure of LSD1 recruitment and accumulation of promoter H3K4 dimethyl marks. Our findings here suggest GFI1 SUMOylation and methylation are part of a series of regulatory inputs that regulate GFI1 function. From these data we propose SNAG domain methylation and linker region SUMOylation coordinate LSD1/CoREST recruitment and enable CoREST dependent activation of LSD1 H3K4 demethylase activity for repression of GFI1 target genes. Our findings add GFI1 to the growing roster of transcription factors regulated by posttranslational modification and provides a rare mechanistic understanding into how these modifications regulate transcription factor functions through the recruitment histone modifying enzyme effectors

The Role of Histone Acetyltransferases in Normal and Malignant Hematopoiesis

Histone or non-histone protein acetylation plays important roles in all kinds of cellular events, including the normal and abnormal development of blood cells, through changing the epigenetic status of chromatin and regulating non-histone protein’s function. Histone acetyltransferases (HATs), which are the enzymes responsible for the histone or non-histone protein acetylation, contain p300/CBP, MYST and GNAT family etc. HATs are not only the protein modifiers and epigenetic factors, but also the critical regulators of cell development and cancerogenesis. Here we will review the function of HATs such as p300/CBP, Tip60, MOZ/MORF and GCN5/PCAF in the normal hematopoiesis and the pathogenesis of hematological malignancies. The inhibitors that have been developed to target HATs will also be reviewed here. Understanding the roles of HATs in normal/malignant hematopoiesis and the underlying mechanism will provide the potential therapeutic targets for the hematological malignancies

Epigenetic dysregulation in chronic myeloid leukaemia: A myriad of mechanisms and therapeutic options

The onset of global epigenetic changes in chromatin that drive tumor proliferation and heterogeneity is a hallmark of many forms cancer. Identifying the epigenetic mechanisms that govern these changes and developing therapeutic approaches to modulate them, is a well-established avenue pursued in translational cancer medicine. Chronic myeloid leukemia (CML) arises clonally when a hematopoietic stem cell (HSC) acquires the capacity to produce the constitutively active tyrosine kinase BCR-ABL1 fusion protein which drives tumor development. Treatment with tyrosine kinase inhibitors (TKI) that target BCR-ABL1 has been transformative in CML management but it does not lead to cure in the vast majority of patients. Thus novel therapeutic approaches are required and these must target changes to biological pathways that are aberrant in CML − including those that occur when epigenetic mechanisms are altered. These changes may be due to alterations in DNA or histones, their biochemical modifications and requisite ‘writer’ proteins, or to dysregulation of various types of non-coding RNAs that collectively function as modulators of transcriptional control and DNA integrity. Here, we review the evidence for subverted epigenetic mechanisms in CML and how these impact on a diverse set of biological pathways, on disease progression, prognosis and drug resistance. We will also discuss recent progress towards developing epigenetic therapies that show promise to improve CML patient care and may lead to improved cure rates

Genomic Imprinting and Cancer: From Primordial Germ Cells to Somatic Cells

Imprinted genes are a subset of genes that are expressed from only one of the parental alleles. The majority of imprinted genes have roles in growth regulation and are, therefore, potential oncogenes or tumour suppressors. Cancer is a disease of aberrant cell growth and is characterised by genetic mutations and epigenetic changes such as DNA methylation. The mechanisms whereby imprinting is maintained in somatic cells and then erased and reset in the germline parallels epigenetic changes that cancer cells undergo. This review summarises what we know about imprinting in stem cells and how loss of imprinting may contribute to neoplasia

Enhancer alterations in cancer: a source for a cell identity crisis

Enhancers are selectively utilized to orchestrate gene expression programs that first govern pluripotency and then proceed to highly specialized programs required for the process of cellular differentiation. Whereas gene-proximal promoters are typically active across numerous cell types, distal enhancer activation is cell-type-specific and central to cell fate determination, thereby accounting for cell identity. Recent studies have highlighted the diversity of enhancer usage, cataloguing millions of such elements in the human genome. The disruption of enhancer activity, through genetic or epigenetic alterations, can impact cell-type-specific functions, resulting in a wide range of pathologies. In cancer, these alterations can promote a ‘cell identity crisis’, in which enhancers associated with oncogenes and multipotentiality are activated, while those promoting cell fate commitment are inactivated. Overall, these alterations favor an undifferentiated cellular phenotype. Here, we review the current knowledge regarding the role of enhancers in normal cell function, and discuss how genetic and epigenetic changes in enhancer elements potentiate oncogenesis. In addition, we discuss how understanding the mechanisms regulating enhancer activity can inform therapeutic opportunities in cancer cells and highlight key challenges that remain in understanding enhancer biology as it relates to oncology