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1.  The era of epigenetics 
Briefings in Functional Genomics  2010;9(5-6):425-428.
doi:10.1093/bfgp/elq039
PMCID: PMC3080744  PMID: 21266345
2.  Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships 
Nucleic Acids Research  2002;30(17):3818-3830.
DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of ∼40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.
PMCID: PMC137423  PMID: 12202767
3.  DNMT1 interacts with the developmental transcriptional repressor HESX1 
Biochimica et biophysica acta  2007;1783(1):131-143.
Hesx1 is a highly conserved homeobox gene present in vertebrates, but absent from invertebrates. Gene targeting experiments in mice have shown that this transcriptional repressor is required for normal forebrain and pituitary development. In humans, mutations in HESX1 impairing either its repressing activity or DNA binding properties lead to a comparable phenotype to that observed in Hesx1 deficient mice. In an attempt to gain insights into the molecular function of HESX1, we have performed a yeast two-hybrid screen and identified DNA methyltransferase 1 (DNMT1) as a HESX1 binding protein. We show that Dnmt1 is co-expressed with Hesx1 within the anterior forebrain and in the developing Rathke’s pouch. Mapping of the interacting regions indicates that the entire HESX1 protein is required to establish binding to a portion of the N-terminus of DNMT1 and its catalytic domain in the C-terminus. The HESX1-DNMT1 complexes can be immunoprecipitated in cells and co-localise in the nucleus. These results establish a link between HESX1 and DNMT1 and suggest a novel mechanism for the repressing properties of HESX1.
doi:10.1016/j.bbamcr.2007.08.010
PMCID: PMC2233781  PMID: 17931718
DNA methylation; Repression; Homeobox; Forebrain; Pituitary; Mouse
4.  DNMT1 interacts with the developmental transcriptional repressor HESX1 
Biochimica et Biophysica Acta  2008;1783(1):131-143.
Hesx1 is a highly conserved homeobox gene present in vertebrates, but absent from invertebrates. Gene targeting experiments in mice have shown that this transcriptional repressor is required for normal forebrain and pituitary development. In humans, mutations in HESX1 impairing either its repressing activity or DNA binding properties lead to a comparable phenotype to that observed in Hesx1 deficient mice. In an attempt to gain insights into the molecular function of HESX1, we have performed a yeast two-hybrid screen and identified DNA methyltransferase 1 (DNMT1) as a HESX1 binding protein. We show that Dnmt1 is co-expressed with Hesx1 within the anterior forebrain and in the developing Rathke's pouch. Mapping of the interacting regions indicates that the entire HESX1 protein is required to establish binding to a portion of the N-terminus of DNMT1 and its catalytic domain in the C-terminus. The HESX1–DNMT1 complexes can be immunoprecipitated in cells and co-localise in the nucleus. These results establish a link between HESX1 and DNMT1 and suggest a novel mechanism for the repressing properties of HESX1.
doi:10.1016/j.bbamcr.2007.08.010
PMCID: PMC2233781  PMID: 17931718
aa, amino acid; DNMT1, DNA methyltransferase 1; SAFB1, scaffold attachment factor beta 1; RNF2, ring finger protein 2; Lonp2, lon peptidase 2, peroxisomal; ZFP592, zinc finger protein 592; BTBD2, BTB (POZ) domain containing 2; SRFBP1, serum response factor binding protein 1; ZMIZ1, zinc finger MIZ-type containing 1; SOD, septo-optic dysplasia; TLE1, transducin-like enhancer of split 1; N-CoR, nuclear co-repressor; eh1, engrailed homology domain 1; GST, glutathione-S-transferase; Gal4DBD, Gal4 DNA binding domain; IVT, in vitro translated; HRP, horse radish peroxidase; PFA, paraformaldehyde; PcP, polycomb group; EZH2, enhancer of zeste homologue; ES cells, embryonic stem cells; DNA methylation; Repression; Homeobox; Forebrain; Pituitary; Mouse
5.  The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase 
Nucleic Acids Research  2003;31(9):2305-2312.
The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modification involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1 enzyme since we find that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1β, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1β associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modifications.
PMCID: PMC154218  PMID: 12711675

Results 1-5 (5)