Epigenetic Modulation of Erythroid Expression: An Integrative Stud of Histone Modifications, Transcription Factors and Transcriptomes.
Open Access
- Author:
- Kumar, Swathi Ashok
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 01, 2012
- Committee Members:
- Ross Cameron Hardison, Dissertation Advisor/Co-Advisor
Robert Paulson, Committee Chair/Co-Chair
David Scott Gilmour, Committee Member
Kateryna Dmytrivna Makova, Committee Member
Francesca Chiaromonte, Special Member - Keywords:
- Epigenetics
erythroid differentiation
multivariate statistics
histone modifications
transcription factors
G1E
G1E-ER4+E2 - Abstract:
- The generation of mature erythroid cells relies on the establishment of lineage-specific differentiation programs that are regulated by transcription factors, co-activators, co-repressors and chromatin modifications. The full extent of this chromatin and transcriptional control, however, has not yet been understood. We explore these questions in a mouse cell line model of erythroid differentiation. G1E is an ES-derived cell line with a genetic knockout of the gene Gata1. G1E cells closely mirror erythroid progenitors BFU-E and CFU-E. An estradiol-dependent rescued subline of G1E (G1E-ER4) mimics normal erythroid maturation on treatment with estradiol. This system provides a physiologically meaningful assay to evaluate genes that are induced or repressed by GATA1. In this thesis, we compared the transcriptomes of G1E and differentiating G1E-ER4 cells to primary progenitors and erythroblasts obtained from HA-tagged fetal liver (E13.4), sorted using Ter119 and CD71. We find that a majority of genes expressed in primary erythroid cells are also expressed in the G1E model, with differentially expressed genes being largely regulated in the same direction and a majority of genes in both systems being repressed. We establish that the transcript profile of G1E cells and primary progenitor, and induced G1E-ER4+E2 cells and primary erythroblasts, are sufficiently similar that meaningful comparisons can be made between transcription factor binding profiles between the primary cells and the G1E cell system. We identify potential long non-coding RNA (lncRNA) in G1E/G1E-ER4+E2. To study the role of transcription factors and histone modifcations in erythroid gene regulation, we comprehensively mapped transcription factors GATA1, GATA2, TAL1 and CTCF, histone modifications H3K4me1, H3K4me3 and H3K27me3, and chromatin accessibility (DHS) and RNA Polymerase II (Pol II), genome-wide, in G1E and induced G1E-ER4 cells. In addition to transcriptome data, from RNAseq, we also obtained the expression profile of mouse genes over a 30 hour time course using the Affemtrix Moe430v2 microarray. We studied the relative impact of the levels and changes in these features on the levels and changes in gene expression using dimension reduction, discriminant analysis and multivariate regression. In both G1E and G1E/G1E-R4+E2, we find that the levels of histone modifications H3K4me3, H3K4me1 and H3K9me3 drive the discriminations between expressed and silent genes with H3K4me1&3 being strongly associated with expression and H3K9me3 being associated with silent genes. We also find that these epigenetic factors do no discriminate between induced and repressed genes in both G1E and G1E-ER4+E2 cell lines. Comparison of the covariation structure of the ten epigenetic features between the G1E and its restored cell line G1E-ER4+E2 revealed limited changes during erythroid differentiation, suggesting that the establishment chromatin marks occurs prior to GATA1-triggered differentiation. We also studied the explanatory power of the epigenetic features in relation of the level of gene expression using regression. Our models explain ~70% of the variation in expression in both progenitor and erythroblast cells. We find that changes in epigenetic features do not significantly increase the power of the discriminatory models. However, a synergy between changes in histone modifications and transcription factor binding contributes to explaining ~35% of the change in expression. We investigate this interplay between transcription factors GATA1, GATA2 and TAL1, in G1E and G1E-ER4+E2. We find that most induced genes have GATA1 and TAL1 close to the start site, while some repressed genes have only GATA1 without TAL1 occupancy. The erythroid transcription factor KLF1 is expressed in primary progenitors and erythroblasts. We find that the EKLF interactome shows very little overlap with regions that bind the erythroid transcription factors GATA1, TAL1 and GATA2 leading to a model that EKLF directs gene expression programs that are independent of those regulated by GATA1 and TAL1.