Date of Award

9-21-2010

Document Type

Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Tae Hoon Kim

Abstract

HOXA CHROMATIN BARRIER CONTAINS CTCF BINDING SITE-DEPENDENT PROMOTER ACTIVITY David Wacker and Tae Hoon Kim. Department of Genetics, Yale University, School of Medicine, New Haven, Connecticut The multifunctional nuclear factor CTCF has been shown to regulate transcription and chromatin structure in three distinct ways. First by directly acting at individual genes as a repressor or an activator to inhibit or stimulate transcription; second, by mediating the interactions of enhancers with nearby promoters via a promoter-enhancer insulating activity; and third, by controlling the boundaries of local chromatin structural domains via an epigenetic barrier activity. Here, we have identified a CTCF binding site that functions as an epigenetic barrier site in the HOXA locus. Using transient transfection assays with reporters constructs, we have determined that the sequence immediately surrounding the CTCF binding site possesses a cell-type-specific promoter activity. This activity is orientation-specific, and is dependent on an intact CTCF binding site. Using Rapid Amplification of cDNA Ends, we have mapped the upstream end of the transcription product, and defined a novel, putative, non-coding RNA with a transcription start site 53 base pairs downstream of the proximal side of the CTCF binding site. We conclude that this is a novel, CTCF binding site-dependent promoter in the HOXA locus, which generates a previously uncharacterized noncoding transcript.

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