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Identification and functional characterisation of novel conserved Hes1 cis-regulatory modules
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Jeziorska, Danuta M. (2011) Identification and functional characterisation of novel conserved Hes1 cis-regulatory modules. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2578181~S1
Abstract
The bHLH transcriptional repressor HES1 plays a pivotal role in progenitor cell
maintenance and cell fate decisions. Hes1 is cyclically expressed during
somitogenesis and in a variety of cell types. In order to better understand the
mechanism of Hes1 expression, we used comparative genomics to identify additional
potential Hes1 cis-regulatory modules (CRMs). This revealed seven phylogenetically
conserved sequence blocks within 57 kb upstream of the Hes1 transcription start site
in mouse. In vitro reporter assays revealed that these regions have a transcriptional
regulatory function as they modulate the activity of both a heterologous and the
endogenous Hes1 promoter. Notch signalling is believed to play a role in Hes1
regulation. Consistent with this, sequence analysis revealed that all of the newly
identified CRMs contain consensus motifs for the RBP-Jĸ Notch effector. ChIP
assays confirmed that RBP-Jĸ binds to all the identified regions in proliferating
C2C12 cells, with the exception of CRM7. Further CRM7 sequence analysis
identified a conserved M-CAT motif which we showed is specifically bound by
TEAD2 and its co-activator YAP using ChIP. The loss of RBP-Jκ or TEAD2 binding
correlated with impaired CRM function. Furthermore, mapping of the chromatin
architecture of the endogenous Hes1 locus showed that several of the identified
CRMs loop onto the Hes1 promoter, consistent with their regulatory function. This
CRM-promoter communication is preserved throughout oscillatory expression of the
gene. These data suggest a molecular mechanism for Notch and Hippo signalling in
Hes1 regulation in proliferating C2C12 cells. Additionally, we have engineered
destabilised Venus fluorescent reporters for live cell imaging of CRM function.
Venus was modified using protein degradation motifs, including the PEST motif
from c-myc and the CL1 yeast sequence, resulting in proteins with half-lives of
approximately 76.5 and 28.7 minutes, respectively. These reporters were
subsequently multimerised using a 2A peptide to enhance their brightness. The
destabilised Venus reporters represent an excellent tool for real-time live cell
imaging.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics | ||||
Library of Congress Subject Headings (LCSH): | Genetic transcription, Gene expression | ||||
Official Date: | June 2011 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Systems Biology Doctoral Training Centre | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Vance, Keith ; Koentges, Georgy | ||||
Sponsors: | Human Frontier Science Program (Strasbourg, France) | ||||
Extent: | xxiii, 191, [7] leaves : ill., charts | ||||
Language: | eng |
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