Disorders of ineffective erythropoiesis cause considerable human morbidity and utilize major health resources. A major barrier to the design of novel treatment strategies is an incomplete understanding of the molecular mechanisms that mediate normal terminal erythroid maturation. The transcription factor GATA-1 is a master regulator of erythropoiesis. In 2004, Weiss and colleagues showed that GATA-1 not only activates many erythroid-specific genes, but also represses nearly an equal number. Subsequent studies showed that both the activated and repressed genes are directly controlled by GATA-1. This has led to two major unanswered questions in the field: (1) how does GATA-1 distinguish between activated and repressed genes? And (2) how does GATA-1 carry out these opposing transcriptional functions? Our long-term obiective is to further elucidate these mechanisms and apply this information to better understand and treat human ineffective erythropoietic disorders. As a first step, we recently performed GATA-1 ChlP-seq and cDNA microarray analysis in murine erythroid cells to identify genome-wide direct functional GATA-1 target genes. This provided a large dataset of activated (454) and repressed (325) GATA-1 target genes and their GATA-1 bound cis-regulatory elements. Bioinformatic analysis revealed candidate features that distinguish GATA-1 activated versus repressed genes. Consistent with the recent work of others, we found that combinatorial occupancy by SCL complexes strongly correlates with gene activation. However, the simple presence of composite GATA:E-box (SCL binding) DNA binding motifs by themselves does not fully distinguish between activated and repressed genes. Therefore, additional information must be required to specify GATA-1 activated genes.
The specific aims of this proposal are to: (1) identify additional factors that cooperate with SCL to distinguish between activated and repressed GATA-1 target genes;and (2) further understand the mechanisms by which SCL co-occupancy results in GATA-1 positive transcriptional activity. Based on our preliminary studies, we hypothesize that certain GC-rich and CAAT binding transcription factors contribute to distinguishing GATA-1 activated genes. We also hypothesize that SCL complexes block interactions between GATA-1 and Polycomb Repressive Complex 2, and recruit positive transcriptional elongation regulators. These hypotheses will be tested using ChlP-seq, gene expression analysis, transgenic reporter assays, and biochemical techniques. The expected outcome from these studies is the identification of novel mechanisms involved in specifying GATA-1 gene activation versus repression.
GATA-1 is a master transcriptional regulator of erythroid development, and is mutated in certain human ineffective erythropoietic disorders. This project is therefore central to the overall theme ofthe program project grant;namely the developmental biology of human erythropoiesis. It dovetails with the other projects on this grant through its study of GATA-1 target genes, its use of complementary approaches/datasets, and its examination of transcriptional elongation regulatory mechanisms in erythroid maturation.
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