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Title: Exploring mechanisms of long range gene regulation at the Pax6 locus
Author: McBride, David J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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A locus-wide DNase I hypersensitive site (HSS) analysis was carried out to identify open chromatin domains at active regulatory elements in cell lines of different origin that do (MV+ and N2A) and do not (RAG) express Pax6. Many HSSs coincided with previously identified regulatory sequences and repetitive DNA elements. Some HSSs are unique to cell line while others were found in all three cell lines. Some of the regulatory elements identified are separated by over 100 kb on the linear DNA molecule. Chromosome Conformation Capture (3C) was used to determine relative crosslinking frequencies of a number of anchor points with a series of DNA fragments spanning the locus, in each of the three cell lines above. Surprisingly, broadly similar levels of association were seen in each cell line, with subtle differences that did not exactly correlate to HSS activity. Findings were mirrored in Pax6-expresing forebrain (14.5 d.p.c.) and cortex cells (17.5 d.p.c.) and Pax6-negative embryonic liver revealing that these associations are not confined to immortalised cell lines. These results suggest a model whereby some regulatory elements do appear to be in close association, but in contrast to results published for other loci, this occurs regardless of whether the elements are active. Despite this, there is some evidence for close interactions between specific cis-elements in cells expressing Pax6 (17.5 d.p.c. Cortex) that are not found in non-expressing cells (RAG). Finally, during this study a series of intergenic transcripts were detected around the Pax6 gene in the region of previously identified distal regulatory elements. These are only present in Pax6 expressing tissues. Preliminary analysis of these transcripts suggests that they do not derive from individually transcribed regulatory elements, but are more likely to be transcribed in one continuous unit.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available