Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Gerd A. Blobel

Abstract

Coordinated transcriptional networks underlie complex developmental processes. Transcription factors play central roles in such networks by binding to core promoters and regulatory elements and thereby controlling transcription activities and chromatin states in the genome. GATA1 is a hematopoietic transcription factor that controls multiple hematopoietic lineages by activating and repressing gene expression, yet the in vivo mechanisms that specify these opposing activities are unknown. By examining the composition of GATA1 associated protein complexes in a genetic complementary erythroid cell system as well as through the use of tiling arrays, we found that a multi-protein complex containing SCL/TAL1, LMO2, Ldb1, and E2A (the SCL complex thereafter) is present at most sites where GATA1 functions as an activator but depleted at most repressive GATA1 sites. Functional interference of the SCL complex selectively impairs activation but not repression by GATA1. These results identify the SCL complex as a critical and consistent determinant of positive GATA1 activity. The SCL complex and GATA1 co-occupy the active &beta-globin promoter and the distant locus control region (LCR), which are juxtaposed into close proximity by chromatin looping. The physical interaction of genes with their cis-regulatory elements are commonly observed in the metazoan genome, however, the molecular architecture of loop formation and the cause-effect relationship between transcription and looping remain unclear. A few lines of evidence suggest Ldb1, one component of the SCL complex, mediates GATA1 induced chromatin looping. To manipulate chromatin loop formation, we employed artificial zinc fingers (ZF) to tether Ldb1 to the &beta-globin promoter in GATA1 null erythroblasts in which the &beta-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the &beta-globin promoter substantially activated &beta-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive in primary erythroblasts with LCR deletion, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation. Our studies have for the first time forced long-range chromatin looping at a native locus and provide a novel approach to manipulate gene activity in cells.

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