Downton, Polly J. (2012) The cis-regulatory module interactome of vertebrate myoD1. PhD thesis, University of Warwick.

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Abstract

Cis,regulatory modules (CRMs) are the functional DNA elements that
encode the spatial and temporal expression patterns of a gene. Each
gene is regulated by multiple CRMs, which may be hundreds of
kilobases distant from the promoter. Many vertebrate CRMs have
been characterised in isolation, but how CRMs act together to regulate
complex patterns of expression is relatively unknown.

Two CRMs that regulate expression of the muscle, specifying master
gene myoD1 have previously been identified. Three additional
potential CRMs were identified using a comparative genomics
approach.The regulatory roles of these CRMs were investigated alone
and in combination. Reporter plasmids containing all thirty,two
possible combinations of these CRMs were made,and their expression
was assayed in an immunologically,defined subpopulation of
transfected mouse myoblast cells by flow cytometry. A statistical
mechanics,based model used this exhaustive expression dataset to
parameterise the interactions between the CRMs. This identified the
ability of particular regulatory modules to have both enhancing and
repressive effects upon transcription, dependent upon their
surrounding CRM ‘context’. The physical proximity of the regulatory modules and myoD1 promoter in native chromatin during gene
expression was confirmed by chromosome conformation capture
analysis. To characterise the molecular basis of these properties
further, a phylogenetic sequence comparison method was used to
identify conserved transcription factor binding sites (TFBSs)within
each CRM. A combination of experimental, bioinformatic and
literature,derived evidence was used to prioritise binding sites fora
large scale mutagenesis study to disentangle the molecular
interactions between a prominent CRM pair. This allowed the
mechanistic underpinnings of context sensitivity in this particular
system to be identified.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH426 Genetics
Library of Congress Subject Headings (LCSH):	Genetic regulation, Developmental genetics, Molecular genetics, Vertebrates -- Genetics
Official Date:	September 2012
Dates:	Date Event September 2012 Submitted
Institution:	University of Warwick
Theses Department:	Systems Biology Doctoral Training Centre
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Koentges, Georgy; Nicodemi, Mario
Extent:	xx, 466 leaves : illustrations, charts
Language:	eng

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