2018 Adams GE PhD.pdf (12.61 MB)
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Understanding the contribution of inositol phosphate signalling to class-1 HDAC complex function

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thesis
posted on 13.11.2019, 11:10 by Grace E. Adams
Class 1 histone deacetylases (HDACs) regulate chromatin confirmation and gene
expression through recruitment to co-repressor complexes. Recently, it was shown
that HDAC3/SMRT binds and is regulated by inositol phosphates (IP) in vitro.
Additionally, complex activity of HDAC1/MTA1 and HDAC1/MIDEAS is enhanced by the
addition of IPs in vitro, indicating conserved regulation. In this work, I aimed to alter
the level of IPs present in the cells through overexpression of a kinase, IPMK, and two
phosphatases, PTEN and SopB, and determine the effect on HDAC activity in vivo. In
addition, I utilised an IPMK knockout embryonic stem (ES) cell line and generated
inducible rescues through a PiggyBac TET system to establish if large scale depletions
of IP levels alter HDAC activity.
We revealed that manipulation of IP through the overexpression of IPMK, PTEN and
SopB does not influence global HDAC activity or histone acetylation levels. Isolation of
overexpressed HDAC3/SMRT in concert with these enzymes, revealed differences in
HDAC3/SMRT complex activity, however, these differences did not correlate with
altered IP levels. Analysis of global HDAC activity, isolated complex activity and HDAC3
target genes in IPMK knockout and rescue ES cell lines further revealed minimal
changes. In conclusion, we were unable to show that IPs regulated the activity of class
I HDAC complexes in vivo. IPMK, HDAC1 and HDAC3 null mice all exhibit early
embryonic lethality suggesting they play essential roles in embryogenesis. Upon
differentiation of TET-IPMK cells, embryoid bodies revealed loss of IPMK leads to
increased cardiomyocyte markers and decreased formation of neuroectoderm
progenitors. Therefore, emphasizing IPMKs important role in gene regulation during
embryogenesis. Our data suggests, that this is not through direct regulation of HDAC
activity, thus highlighting an undiscovered nuclear role for IPMK.

History

Supervisor(s)

Shaun Cowley; John Schwabe

Date of award

04/05/2018

Author affiliation

Department of Molecular and Cell Biology

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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