Role of Zinc Finger Protein WIZ in the recruitment of Histone Methylase G9a
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Özkan2017.pdf (8.611Mb)
Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Özkan, Burak
Metadata
Abstract
The N-terminal tails of histones are subject to many chemical modifications that are
involved in a variety of biological functions. Histone methylation is a major epigenetic
modification found in both single and multicellular organisms and is directly involved in
the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been
shown to have diverse functions depending on the number of methyl groups added;
H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within
inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic
histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses
addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to
silence genes. Therefore, it is a vital component of the gene expression regulation
machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the
G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger
protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core
G9a complex, which is essential for mouse development. Lack of any G9a complex
member leads to embryonic lethality at E9.5 with severe growth defects.
The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro
(Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with
H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a
complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood.
Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a
complex to specific sites must be mediated by other binding partners of the G9a complex.
Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding
partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and
GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The
aim of this study was to determine the role of WIZ in the recruitment of the G9a complex
to specific sites. I showed that knockdown of WIZ had no significant effect on the
chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is
dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the
G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising
G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc
finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands
exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ
preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with
synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain
distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with
higher affinity than dsDNA, and binding to ssDNA is sequence-independent.
This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and
RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ
during differentiation. Furthermore, DNA binding preference of WIZ might suggest that
WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops
where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive
sequences.