Xinsheng Nan

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 (ref. 5) and MeCP2 (ref. 6) 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.

MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin

MeCP2 is an abundant mammalian protein that binds to methylated CpG. We have found that native and recombinant MeCP2 repress transcription in vitro from methylated promoters but do not repress nonmethylated promoters. Repression is nonlinearly dependent on the local density of methylation, becoming significant at the density found in bulk vertebrate genomic DNA. Transient transfection using fusions with the GAL4 DNA binding domain identified a region of MeCP2 that is capable of long-range repression in vivo. Moreover, MeCP2 is able to displace histone H1 from preassembled chromatin that contains methyl-CpG. These properties, together with the abundance of MeCP2 and the high frequency of its 2 bp binding site, suggest a role as a global transcriptional repressor in vertebrate genomes.

The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation.

DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys9methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.

Purification of CpG islands using a methylated DNA binding column

CpG islands are short stretches of DNA containing a high density of non–methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl–CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full–length cDNAs and to place genes on genomic maps.

DNA methylation specifies chromosomal localization of MeCP2.

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

Interaction between chromatin proteins MECP2 and ATRX is disrupted by mutations that cause inherited mental retardation

Mutations in the human methyl-CpG-binding protein gene MECP2 cause the neurological disorder Rett syndrome and some cases of X-linked mental retardation (XLMR). We report that MeCP2 interacts with ATRX, a SWI2/SNF2 DNA helicase/ATPase that is mutated in ATRX syndrome (α-thalassemia/mental retardation, X-linked). MeCP2 can recruit the helicase domain of ATRX to heterochromatic foci in living mouse cells in a DNA methylation-dependent manner. Also, ATRX localization is disrupted in neurons of Mecp2-null mice. Point mutations within the methylated DNA-binding domain of MeCP2 that cause Rett syndrome or X-linked mental retardation inhibit its interaction with ATRX in vitro and its localization in vivo without affecting methyl-CpG binding. We propose that disruption of the MeCP2–ATRX interaction leads to pathological changes that contribute to mental retardation.

Transcriptional repression by methylation of CpG

Summary Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.

Regulation of MBD1-mediated transcriptional repression by SUMO and PIAS proteins

In mammalian cells, DNA methylation is associated with heritable and stable gene repression, mediated in part by methyl‐CpG‐binding domain (MBD) proteins that recruit corepressors to modify chromatin. MBD1 protein, a member of the MBD family, forms a complex with SETDB1 histone methylase to silence transcription at target promoters by methylation of lysine 9 of histone H3. How MBD1‐mediated transcriptional repression is regulated is currently unknown. Here we show that MBD1 is a target for sumoylation by PIAS1 (Protein Inhibitors of Activated STAT 1) and PIAS3 E3 SUMO (small ubiquitin‐like modifier)‐ligases, at two conserved lysine residues within the C‐terminus of MBD1. Although sumoylated MBD1 binds to methylated DNA, it does not incorporate into a complex with SETDB1 and does not efficiently repress transcription of a target gene, p53BP2, in HeLa cells. Our data suggest that transcriptional silencing by MBD1 is regulated by a PIAS‐mediated conjugation of SUMO1, which antagonizes the formation of a repressive complex with SETDB1.

Studies of DNA méthylation in animals

SUMMARY We have been studying the evolution and function of DNA methylation in vertebrate animals using three related approaches. The first is to further characterise proteins that bind to methylated DNA. Such proteins can be viewed as ‘receptors’ of the methyl-CpG ‘ligand’ that mediate downstream consequences of DNA modification. The second approach involves CpG islands. These patches of non-methylated DNA coincide with most gene promoters, but their origin and functional significance have only recently become the subject of intensive study. The third approach is to trace the evolution of DNA methylation. Genomic methylation patterns of vertebrates are strikingly different from those of invertebrates. By studying methylation in animals that diverged from common ancestors near to the invertebrate/vertebrate boundary, we will assess the possibility that changes in DNA methylation contributed causally to the evolution of the complex vertebrate lineage.

Doublesex and mab-3–related transcription factor 5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral-medial progenitor fate

Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3–related transcription factor 5 (Dmrt5) as a marker for mammalian ventral-medial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain- and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventral-medial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.