Disease-associated Brd4 mutation – linking chromatin binding and the DNA damage response
View/ Open
Date
06/07/2019Author
Olley, Gabrielle Jade
Metadata
Abstract
Acetylation of lysine residues is a histone modification associated with active chromatin. The
modified residues provide docking sites for the epigenetic reader BRD4, which binds to the
acetylated lysines via its two bromodomains. BRD4 is known to be involved in RNA
polymerase II activation, maintaining the pluripotency of embryonic stem cells and in DNA
damage response signalling.
In this thesis, I describe a newly identified missense mutation (Y430C) in BRD4 in a patient
with a Cornelia de Lange syndrome (CdLS)-like phenotype. CdLS is a neurodevelopmental
disorder that can cause a range of symptoms including upper limb malformations,
craniofacial abnormalities and intellectual disability and is usually associated with mutations
in components of the cohesin complex and the cohesin loader NIPBL. How these mutations
may cause CdLS is currently unknown, but the assumption has been that they cause altered
gene regulation during development.
Using a mouse embryonic stem cell (mESC) line, engineered through CRISPR-Cas9
technology to be homozygous for the patient mutation in BRD4, I show that the mutation
decreases the affinity of BRD4 for acetylated lysines. This causes a loss of BRD4 binding in
the genome, most noticeable at cis-regulatory elements. However, surprisingly I found no
evidence for altered gene expression in the cells with the Brd4 mutation. Instead I identify
increased G2/M checkpoint activation in the Y430C mESCs compared to wild-type cells,
and my research suggests that this is attributable to an increase in DNA damage signalling