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Cis-regulatory elements of the Hoxc11 gene

Cis-regulatory elements of the Hoxc11 gene
Cis-regulatory elements of the Hoxc11 gene

To fill gaps in our knowledge about the functional roles of posterior Hoxc genes, in this thesis I set out to identify and characterise cis-regulatory elements contributing to their regulation.

The starting point was a l0kb region of DNA local to the Hoxc11 transcription unit, presumed to harbour enhancer elements based on transgenic reporter gene evidence.  In order to identify these elements, phylogenetic footprinting was performed initially utilizing genomic sequence comparisons between mouse and human sequences.  Due to the slowly evolving nature of the Hox clusters, extremely stringent parameters for the sequence comparisons had to be developed in order to avoid false positive Conserved Noncoding Sequence elements (CSEs), while still filtering out CSEs with regulatory function (avoidance of false negatives).  Accordingly, the cut-off criteria were set at 97% similarity over a 100bp window of comparison, and three CSEs where identified in the local Hoxc11 region, namely R1, R2, and R3. 

A series of Hoxcll/1acZ reporter gene deletion constructs was designed where deletion breakpoints were based on positions of R1, R2, and R3.  In vivo recombination was used to create these deletions with pinpoint accuracy.  However, none of these Hoxc11 constructs showed appreciable activity in a number of cell lines despite confirmation of expression for the endogenous homolog.  While this raised the possibility of an incompatibility between the Hoxc11 promoter and the local elements, a series of constructs was launched to investigate enhancer activity per se of elements R1, R2, and R3 by combining them with a heterologous promoter and luciferase reporter.  This demonstrated that element R3 could significantly enhance transcription from the minimal SV40 promoter in CaCo-2 cells, and to a lesser degree in HeLa cells.

University of Southampton
Blumer, Zoë Danielle
Blumer, Zoë Danielle

Blumer, Zoë Danielle (2005) Cis-regulatory elements of the Hoxc11 gene. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

To fill gaps in our knowledge about the functional roles of posterior Hoxc genes, in this thesis I set out to identify and characterise cis-regulatory elements contributing to their regulation.

The starting point was a l0kb region of DNA local to the Hoxc11 transcription unit, presumed to harbour enhancer elements based on transgenic reporter gene evidence.  In order to identify these elements, phylogenetic footprinting was performed initially utilizing genomic sequence comparisons between mouse and human sequences.  Due to the slowly evolving nature of the Hox clusters, extremely stringent parameters for the sequence comparisons had to be developed in order to avoid false positive Conserved Noncoding Sequence elements (CSEs), while still filtering out CSEs with regulatory function (avoidance of false negatives).  Accordingly, the cut-off criteria were set at 97% similarity over a 100bp window of comparison, and three CSEs where identified in the local Hoxc11 region, namely R1, R2, and R3. 

A series of Hoxcll/1acZ reporter gene deletion constructs was designed where deletion breakpoints were based on positions of R1, R2, and R3.  In vivo recombination was used to create these deletions with pinpoint accuracy.  However, none of these Hoxc11 constructs showed appreciable activity in a number of cell lines despite confirmation of expression for the endogenous homolog.  While this raised the possibility of an incompatibility between the Hoxc11 promoter and the local elements, a series of constructs was launched to investigate enhancer activity per se of elements R1, R2, and R3 by combining them with a heterologous promoter and luciferase reporter.  This demonstrated that element R3 could significantly enhance transcription from the minimal SV40 promoter in CaCo-2 cells, and to a lesser degree in HeLa cells.

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Published date: 2005

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Local EPrints ID: 465981
URI: http://eprints.soton.ac.uk/id/eprint/465981
PURE UUID: e0c85a70-eee4-4f32-8f65-66fbf4c37631

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Date deposited: 05 Jul 2022 03:52
Last modified: 05 Jul 2022 06:41

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Author: Zoë Danielle Blumer

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