Download Understanding the role of HDAC1 in transcriptional activation

Potential First Supervisor name:
Affiliation:
Dr Shaun Cowley
Department of Biochemistry,
University of Leicester
Potential Second Supervisor name:
Affiliation:
Dr Ralf Schmid
Department of Biochemistry,
University of Leicester
Potential Supervisor name: n/a
Affiliation: n/a
PhD project title:
Understanding the role of HDAC1 in transcriptional activation
University of Registration: Leicester
Project outline, timing, and budget
1. Project outline describing the scientific rationale of the project (max 4,000 characters incl.
spaces and returns)
Introduction – Histone deacetylase 1 and 2 (HDAC1/2) are sister proteins (82% amino
acid identity) with essential functions in transcription, DNA repair, DNA synthesis and
mitosis [1]. Biochemically, their role is to modulate levels of lysine-acetylation (Lys-Ac), a
dynamic post-translational modification which occurs on approximately 1,750 proteins [2].
The levels of Lys-Ac are determined by the opposing actions of lysine acetyltransferases
and HDAC enzymes. In the context of histones and chromatin, acetylation results in a more
‘open’ conformation, in which the DNA is more accessible and transcriptionally permissive;
HDACs therefore have been implicated in the process of transcriptional repression.
However, recent ChIP-seq studies suggest that HDAC1 is predominantly located at ‘active’
genes in cells, suggesting the requirement for cyclical acetylation and deacetylation of
histones during transcriptional activation. We intend to study the role of HDAC1 in
transcriptional activation using a combination of wet-lab ‘genome editing’ (Cowley lab) and
in silico comparative transcriptome analysis (Schmid lab).
Basic Bioscience underpinning health:
As enzymes, HDAC1/2 make excellent therapeutic targets. Drugs which inhibit their activity
cause cells to exit cell cycle and undergo apoptosis, prompting their use as anti-cancer
agents. However, despite their promise, we still do not fully understand how inhibition of
HDAC1/2 inhibits cellular proliferation.
Research question – What is the molecular basis of HDAC1’s role in gene activation?
Experimental Approach: We recently showed that double deletion of Hdac1 and Hdac2 in
embryonic stem (ES) cells leads to massive cell death 4 days after gene inactivation, due to
a deregulated transcriptome and an inability to accurately segregate chromosomes during
mitosis (Jamaladdin et al, 2014 PNAS vol. 111 no. 27 9840–9845). This study also
confirmed that a reduction in the level of HDAC in the cell correlated with increased severity
of the chromosome segregation phenotype. It would therefore be advantageous to be able
to control the level of endogenous HDAC1, in the absence of HDAC2, so that we could
produce just enough protein to avoid cell death while but were still able to study the gene
expression and differentiation phenotypes.
In order to achieve this we aim to combine two
technologies: 1) ‘ProteoTuner’ - the addition of a
‘destruction domain’ from a mutated FKBP12 protein,
which makes the stability of the protein of interest
controllable by the addition or removal of a ligand,
shield-1 [3].
2)
CRISPR genome editing
methodologies (the nuclease Cas9 + a guide-RNA)
allow the addition of new DNA to almost any genomic
locus [4]. We aim to add the destruction domain (DD)
sequence (110 amino acids) to both copies of the
Hdac1 gene in Hdac2 conditional knockout cells (see
Figure 1). This will allow us to inactivate HDAC2
(using Cre-ER, as in our previous studies) and then
regulate the level of HDAC1 protein using different
concentrations of shield-1.
A
B
Cas9
gRNA
FRT
FRT
HDAC1
DD
Flag
Neo
HDAC1
DD
Flag
Hyg
Figure. 1 Strategy to create an ES cell line in which both
copies of Hdac1 gene have been tagged with a ‘destruction
domain’ (DD). A) Addition of a destruction domain (DD –
110 amino acids) to a protein of interest (POI) allows its level
to regulated (stabilized) by the addition by the ligand,
shield-1. B) CRISPR mediated gene-targeting will be used to
simultaneously add tags to both Hdac1 alleles using double
drug selection (Neo + Hyg).
Goals:
•
•
•
•
Characterize Hdac1DD/DD ; HDAC2Lox/Lox; Cre-ER embryonic stem cells, initially by titrating
the amount of shield-1 to give the desired low level of HDAC1 (HDAC1Low) while still
retaining cell viability.
HDAC1 is historically thought to play a role in gene repression, however recent ChIP-seq
data suggests that it is predominantly located at ‘active’ genes. We will therefore
monitor gene activation via RNA-seq in HDAC1low cells following 2 hour RA treatment.
The kinetics of gene activation will be examined e.g. RNA pol II recruitment and general
transcription factors by chromatin IP followed by next-Gen sequencing (ChIP-seq).
Bioinformatic analysis of the RNA-seq and ChIP-seq data (performed in the Schmid lab)
will be used to compare the transcriptome and ChIP-seq data from HDAC1Low cells to
publically available data sets in the Geo Expression Omnibus (GEO) and ArrayExpress
databases to identify proteins/inhibitor treatments which regulate similar transcriptional
networks.
References:
1. Kelly RD, Cowley SM (2013) The physiological roles of histone
deacetylase (HDAC) 1 and 2: Complex co-stars with multiple leading parts. Biochem Soc
Trans 41(3):741–749. 2. Choudhary, C. et al. Science 325, 834-840, (2009). 3.
Banaszynski et al, Cell 126, 995–1004, (2006). 4. Ran et al, Nature Methods VOL.8
NO.11 2281- 2308 (2013).
Please list the techniques that will be undertaken during the project.
Stem cell culture, design and use of CRISPR gene editing to generate knock-out and
tagged cell lines, Southern blotting, western blotting, qRT-PCR, immunofluorescence,
immunoprecipitation of HDAC containing complexes from mammalian cells, ChIP, FACS,
RNA-seq, ChIP-seq, bioinformatic and statistical analysis of RNA-seq and ChIP-seq
datasets.