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Title: Identification and functional analysis of SCL and LYL1 regulatory elements
Author: Chapman, M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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The SCL gene is a critical haematopoietic transcription factor, with a pattern of expression that is highly conserved from mammals to teleost fish. Its central role in haematopoiesis and its complex expression pattern make it a model gene for studying transcriptional networks. The full set of cis-regulatory elements active in the SCL locus is probably still to be discovered, and will be required for a more complete understanding of its transcriptional regulation. SCL has also been a model gene for studying comparative sequence analysis. This has been exploited in this thesis to approach two questions. Firstly: how can different species be utilized and combined to improve the accuracy of comparative sequence analysis? Secondly: what previously undiscovered cis-regulatory elements are active in the SCL locus, and how do they act? BAC clones containing the SCL loci of dog and platypus as well as the paralogous LYL1 locus of a marsupial, the stripe-faced dunnart, were isolated and sequenced and the SCL locus in rat was annotated. Human/dunnart sequence comparisons appeared to be more selective than human/mouse comparisons in the LYL1 locus, but still permitted the identification of all putative promoters within the clone. Phylogenetic footprinting determine five transcription factor binding sites within the LYL1 promoter, with a pattern very reminiscent of those seen in critical SCL regulatory elements. Functional analyses confirmed LYL1 promoter activity and nuclear binding. Comparisons of two-, three-, and four-way eutherian alignments at the SCL locus revealed that the four-way alignment was the most sensitive and specific for identifying both regulatory regions and transcription factor binding sites. Adding platypus sequence vastly improved specificity, but at the expense of sensitivity. Four candidate regulatory regions was identified, three of which were then shown to act as lineage-restricted regulatory elements, probably affecting transcription of the co-ordinately expressed downstream gene, MAP17. It is concluded that to maximize sensitivity and specificity in the identification of eutherian regulatory regions, sequences from multiple eutherian species should be combined for comparative analysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available