MAPPING, CONSERVATION AND FUNCTION OF BINDING SITES FOR ERTHYROID TRANSCRIPTION FACTOR GATA1 IN THE MOUSE GENOME
Open Access
- Author:
- Cheng, Yong
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 28, 2009
- Committee Members:
- Ross Cameron Hardison, Dissertation Advisor/Co-Advisor
Ross Cameron Hardison, Committee Chair/Co-Chair
Robert Paulson, Committee Member
Stephan C Schuster, Committee Member
David Scott Gilmour, Committee Member
Reka Z Albert, Committee Member - Keywords:
- conservation
ChIP-chip
GATA1
function - Abstract:
- Precise control of gene expression in time and space is required for normal tissue development and function. Transcription factors play an important role in regulating gene expression. Thus, identifying the binding sites of transcription factors in vivo and understanding their function is a major goal of molecular genetics and biochemistry, and now obtaining this information in a genome-wide manner is a goal of functional genomics. In this thesis, we use erythoroid cells and GATA1 as our model system to study the connection between transcription factors (e.g. GATA1) and gene regulation. Erythropoeisis is the process by which erythrocytes are produced. GATA1 is an erythroid transcription factors that is essential for this process. In the first study reported in this thesis, we examined the relationship between evolutionary preservation of a transcription factor binding site motif and its activity. We used chromatin immunoprecipitation combined with a high density tiling array of a 66 megabase (Mb) region of mouse chromosome 7 (ChIP-chip) to identify DNA segments occupied by GATA1 . We then tested each occupied segment for enhancer activity in a transient transfection assay to identify the GATA1-occupied segments with this function. Combining these functional analyses with the constraint information of the consensus GATA1 binding motif (WGATAR) in the occupied segments, we found that GATA1-bound segments with high enhancer activity tend to be the ones with an evolutionarily preserved WGATAR motif. This relationship was confirmed by a loss-of-function assay by mutating each individual WGATAR motif and measuring its contribution to the whole enhancer activity. Thus, GATA1 binding site motifs that regulate gene expression during erythroid maturation are under strong selective constraint, while non-constrained binding may have only a limited or indirect role in regulation. After this analysis of the relationship between motif constraint and activity (in the 66 Mb regions), we then examine the relationship between occupancy and expression response genome-wide. We mapped the GATA1-occupied segments in the whole mouse genome using NimbleGen HD2 whole-genome tiling array. We also obtained the expression profile of mouse genes in response to restoring GATA1, using the Affemtrix Moe430v2 microarray. Our results showed strong correlation between GATA1 occupancy pattern around the transcription start site (TSS) of genes and the expression pattern of the corresponding genes. All responsive genes tend to be closer to GATA1-occupied segments than randomly chosen genes, and this relationship is more pronounced for up-regulated genes than down-regulated genes. Up-regulated genes also tend to have multiple GATA1-occupied segments in their vicinity, with a substantial signal for occupancy close to the TSS and in the first intron. These positional relationships that tend to distinguish up- from down-regulated genes indicate different mechanisms of regulation. Most up-regulated genes have GATA1 and other activating co-factors close to the start site. Some down-regulated genes have GATA1 but not other activating transcription factors such as TAL1. Other down-regulated genes may be repressed by an indirect mechanism, e.g. as a result of the activation of other genes.