Functional characterization of the DNA glycosylase; Methyl-‐CpG binding domainpProtein 4 (MBD4)
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Date
06/07/2013Author
Meng, Huan
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Abstract
DNA methylation is a major form of epigenetic modification and involves the addition of a
methyl group covalently to the 5-position of the cytosine pyrimidine ring, mostly within the
context of CpG dinucleotides in vertebrate somatic cells. Methylation of CpG dinucleotides
at promoter regions is generally associated with transcriptional repression. In this context,
the methyl-CpG binding proteins (MeCPs) that are capable of recognition of methylated
CpG dinucleotides are proposed to play a central role in DNA methylation associated
transcriptional repression. Methyl-CpG binding domain protein 4 (MBD4) is an MeCP that
possesses a glycosylase domain at its C-terminal, which can excise and repair both G:T and
G:U mutations derived from DNA deamination at CpG dinucleotides, in addition to its Nterminal
MBD binding domain. MBD4 has been associated with a number of pathways
including DNA repair, apoptosis, transcriptional repression, and possibly DNA
demethylation processes. However, the precise contribution of MBD4 to these processes
remains unclear.
To explore the functional repertoire of MBD4 I decided to undertake multiple
protein interaction studies to identify potential partner proteins. I performed yeast 2-hybrid
screens with an 11.5 day mouse embryonic cDNA library and multiple mass spectrometry of
immunoprecipitates of tagged versions of MBD4 that were over-expressed in human cell
lines. I detected ~380 potential interacting candidates with these assays. A significant
number of candidates were detected in both assay systems. Chosen candidates were further
validated by reciprocal co-IP of expressed partners and by immunofluorescence (IF)
microscopy to determine their potential co-localisation in mouse and human cell lines.
Subsequently, I identified the intervening domain of MBD4 as a novel protein interaction
region for tested candidates. My analysis suggests that MBD4 can have a role in regulation
of post-replication methyl-error repair/methylation machinery through its direct interaction
with DNMT1 (previously shown), UHRF1 (novel) and USP7 (novel), as well as possible
cross-talk to histone modification and chromatin remodelling pathways, through partners
such as PRMT5 and ACF1. Interestingly the transcription regulatory components KAP1 and
CFP1 not only interact with but also dramatically influence the stability of exogenously
expressed MBD4 in human cells. In general positive validation by IP and IF demonstrates
the robustness of the initial screens, and implies that MBD4 may impact upon several
transcriptional and epigenetic networks along with a number of nuclear pathways that
include transcriptional repression, DNA repair and RNA processing.
To test for transcriptional aberration in the absence of Mbd4 function I profiled two
independent mouse cell lines that lack MBD4 activity using Illumina MouseWG-6 v2.0
Expression BeadChip arrays. A number of genes were identified that are significantly up- or
down- regulated in both Mbd4-/- MEFs. This included mis-expression of insulin-like growth
factor-binding proteins and two paternally imprinted genes Dio3 and H19. The cohort of
genes that were mis-expressed in the Mbd4-/- MEFs overlap with genes that responsed to
tamoxifen exposure in an ER-positive ZR-75-1 xenograft model. In response to this
observation I identified a potential interaction between MBD4 and estrogen receptor α (ERα)
by co-IP and IF co-localisation. This suggests that MBD4 might potentiate transcription of
estrogen regulated genes via a direct interaction with ERα, supporting a possible link
between replication repair remodelling and steroid/thyroid hormone receptor transcriptional
regulation. Additionally I performed a pathway analysis by which several developmental
genes including Sox9, Klf2 and Klf4, were prioritised as possible MBD4 targets. On this
basis I propose a role for MBD4 in acquired diseases such as cancers and autoimmune
diseases via transcriptional regulation.
I also performed a comparison of MBD4 DNA binding activity with MBD4
homologues from the Medaka fish (Oryzias latipes) and the amphibian, Xenopus laevis. I
could show that DNA binding specificity to a series of methylated and mismatched probes is
conserved regardless of the poor sequence conservation of the MBD domain of MBD4
between the species. I conclude that MBD4 is integrated in multiple pathways in the nucleus
that includes DNA repair, chromatin remodelling, transcriptional regulation and genome
stability.