Roles of histone modifications and transcription factors in transcription regulation in erythropoiesis

Cellular differentiation is a process in which pluripotent cells become morphologically and functionally more specialized cells, which happens in the development from monocellular zygote into a multicellular organism system with complex cells and tissues of different types, and also in cell turnover or renewal in adults. This process is largely driven by differential expression of genes so that they are expressed in appropriate temporal and quantitative manners for specific production of proteins that characterize the committed cell type. The output of specific gene expression profiles are substantially controlled by the relevant regulation of gene transcription. Bulk of the studies of gene transcription regulation mechanisms fall in the area of epigenetics which looks into biochemical events that occur without changing the genomic DNA sequence but often result in alterations of gene expression. The main epigenetic events include histone modifications, binding of cis-regulatory elements by transcription factors, chromatin remodeling and DNA methylation. In this thesis, we investigated the distributions of four critical histone modifications, DNase hypersensitivity, and binding sites of three important transcription factors on mouse genome in mouse G1E model which mimics normal erythroid differentiation, allowing us to study the relationship between these epigenetic features and specific gene regulation in erythropoiesis, which is a well-defined cellular differentiation process for maturation of red blood cells.;As described in the second chapter of this thesis, we applied ChIP-seq technique to examine the genome-wide distributions of four histone modifications in both erythroid progenitor G1E cells and differentiating erythroblast G1E-ER4+E2 cells. The modifications are H3K4me3 which is associated with promoters, H3K4me1 which marks enhancers, H3K27me3 which associates with gene repression, and H3K9me3 which is associated with heterochromatin. We employed a multivariate HMM to segment the genome into six chromatin states defined by the different distributions of the examined histone modifications. Comparison of the chromatin states between the G1E and its restored cell line G1E-ER4+E2 revealed quite limited alterations of the states in the captured erythroid differentiation process, suggesting a preceding establishment of the chromatin states prior to the GATA1-triggered differentiation, most likely at the stage of erythroid lineage commitment.;As a parallel project to the study of histone modifications, we utilized the same ChIP-seq approach and the same G1E system to identify the genome-wide localization of occupancy by three critical transcription factors and investigated their roles in transcription regulation in erythroid differentiation. These factors are GATA1 which is a master regulator of erythropoiesis, TAL1 which is also essential to the lineage and often co-binds DNA with GATA1, and GATA2 which is important for cell pluripotency maintenance in earlier hematopoietic stages. Our observations confirmed the replacement of GATA2 by GATA1, co-occupancy of GATA1 and TAL1, and its association with gene induction in erythropoiesis on a genome-wide scale. Our examination of the motif distribution on TAL1 binding sites suggested TAL1 localization is substantially controlled by GATA1 binding. And our investigation of distributions of H3K4me1 and H3K27me3 on GATA1 binding sites attributed them as strong determinant in GATA1 occupancy prediction. In addition, an enhancer assay of two predicted GATA1 motif containing CRMs proved one of them to be an enhancer, which is bound by GATA1 to up-regulate the transcription of a downstream microRNA locus, supporting microRNA as a mechanism mediating transcription regulation by GATA1.