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H H Ng

Publications and source records attributed to H H Ng.

10 recordsLinked to original sources
PubMed2000

Histone deacetylases: silencers for hire.

Over the past few years, the long-standing idea that covalent modification of chromatin can play a role in determining states of gene activity has been confirmed. Eukaryotic genes can be silenced by deacetylation of acetyl-lysine moieties in the N-terminal tails of histones. Recent work links histone deacetylases with an increasing number of repressors, suggesting that deacetylation might be a rather pervasive feature of transcriptional repression systems.

Animals↗
PubMed2000

Active repression of methylated genes by the chromosomal protein MBD1.

MBD1 belongs to a family of mammalian proteins that share a methyl-CpG binding domain. Previous work has shown that MBD1 binds to methylated sites in vivo and in vitro and can repress transcription from methylated templates in transcription extracts and in cultured cells. In the present study we established by several experimental criteria that, contrary to a previous report, MBD1 is not a component of the MeCP1 repressor complex. We identified a powerful transcriptional repression domain (TRD) at the C terminus of MBD1 that can actively repress transcription at a distance. Methylation-dependent repression in vivo depends on the presence of both the TRD and the methyl-CpG binding domain. The mechanism is likely to involve deacetylation, since the deacetylase inhibitor trichostatin A can overcome MBD1-mediated repression. Accordingly, we found that endogenous MBD1 is particularly concentrated at sites of centromeric heterochromatin, where acetylated histone H4 is deficient. Unlike MBD2 and MeCP2, MBD1 is not depleted by antibodies to the histone deacetylase HDAC1. Thus, the deacetylase-dependent pathway by which MBD1 actively silences methylated genes is likely to be different from that utilized by the methylation-dependent repressors MeCP1 and MeCP2.

Acetylation↗
PubMed1999

The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites.

In addition to its well-documented effects on gene silencing, cytosine methylation is a prominent cause of mutations. In humans, the mutation rate from 5-methylcytosine (m5C) to thymine (T) is 10-50-fold higher than other transitions and the methylated sequence CpG is consequently under-represented. Over one-third of germline point mutations associated with human genetic disease and many somatic mutations leading to cancer involve loss of CpG. The primary cause of mutability appears to be hydrolytic deamination. Cytosine deamination produces mismatched uracil (U), which can be removed by uracil glycosylase, whereas m5C deamination generates a G x T mispair that cannot be processed by this enzyme. Correction of m5CpG x TpG mismatches may instead be initiated by the thymine DNA glycosylase, TDG. Here we show that MBD4, an unrelated mammalian protein that contains a methyl-CpG binding domain, can also efficiently remove thymine or uracil from a mismatches CpG site in vitro. Furthermore, the methyl-CpG binding domain of MBD4 binds preferentially to m5CpG x TpG mismatches-the primary product of deamination at methyl-CpG. The combined specificities of binding and catalysis indicate that this enzyme may function to minimize mutation at methyl-CpG.

Amino Acid Sequence↗
PubMed1999

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.

Adenosine Triphosphatases↗
PubMed1999

DNA methylation and chromatin modification.

DNA methylation and chromatin modification are two global mechanisms that regulate gene expression. Recent studies provide insight into the mechanism of transcriptional silencing by a methyl-CpG binding protein, MeCP2. MeCP2 is shown to interact with the Sin3/histone deacetylase co-repressor complex. Thus, this interaction can provide a mechanistic explanation for the long-known relationship between DNA methylation and chromatin structure. Moreover, several studies have shown that inhibition of histone deacetylases by specific inhibitors can reactivate endogenous genes or reporter constructs previously silenced by DNA methylation. Taken together, the data strongly suggest that DNA methylation can pattern chromatin modification.

Acetylation↗
PubMed1999

MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex.

Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes.

Amino Acid Sequence↗
PubMed1998

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.

Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

Acetylation↗
PubMed1997

Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1.

DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells.

Amino Acid Sequence↗
PubMed1996

Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs.

We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.

Amino Acid Sequence↗