Solution structure of the matrix attachment region-binding domain of chicken MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel beta-sheet and an alpha-helix alpha1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, alpha2/alpha3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein.DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.     

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.     

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.     

DNA binding of methyl-CpG-binding protein MeCP2 in human MCF7 cells

MeCP2 has been identified as a chromatin-associated protein that recognizes MAR elements as well as methyl-CpGs. To characterize target sequences of MeCP2 in human cells, we employed two complementary methods. First, by use of a preparative chromatin immunoprecipitation protocol, we created from MCF7 cells a library enriched with sequences bound to MeCP2. A total of 154 representative clones were sequenced and analyzed. A large fraction of clones was found to be associated with retrotransposons, mostly with Alu repeats. A subgroup of four clones is derived from putative MARs; one clone is associated with a CpG island, and four clones contain alphoid repeats. Classical satellite DNAs II and III are not represented among clones, although they are heavily methylated. Second, using indirect immunofluorescence microscopy, we show that MeCP2 staining of human metaphase chromosomes has a dotted to knobby appearance with a reduced level of staining of centromeric regions of some chromosomes. On the other hand, an anti-5-methylcytosine antibody preferentially stained the juxtacentromeric regions of chromosomes 1, 9, and 16, which habor highly methylated, classical satellite DNAs, and methylated alphoid sequences in centromeric regions of several other chromosomes with reduced intensity. In interphase MCF7 cells, the distribution of MeCP2 exhibits a granular appearance throughout the nucleus. This distribution does not parallel that of methylated cytosine and heterochromatin. The selective binding behavior of MeCP2 revealed by these results (preference for murine major satellite DNA, Alu sequences, MARs, and CpG islands) is explained by its ability to recognize the sequence information (guanine bases) adjacent to CpG (TpG) as demonstrated in previous footprinting experiments.     

Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.     

Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure

The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1^−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function.     

Engineering a high-affinity methyl-CpG-binding protein

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.     

Regulation of mouse satellite DNA replication time.

The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.     

Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.     

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brown^Dominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.     

Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein

An increase in the dose of the Su(var)3-7 locus of Drosophila augments heterochromatin-promoted variegated silencing. The deduced protein sequence of Su(var)3-7 reveals seven widely spaced zinc fingers. We found that Su(var)3-7 has affinity for DNA in vitro and that the minimal protein sequence requirement for DNA binding is any module containing two zinc fingers and the interval between them. As Su(var)3-7 is a heterochromatin-associated protein, we tested its affinity for various satellite DNA sequences in vitro. The AATAT and 353-bp elements have the highest affinity. If affinity for satellite DNAs contributes to the presence of Su(var)3-7 in heterochromatin, a general affinity for DNA, or sequences yet to be determined, suggests a function in the genomic silencing of position-effect variegation: expansion of heterochromatin, whether continuous by spreading or discontinuous by pairing with sequence elements scattered through euchromatin, could use the affinity of Su(var)3-7 for DNA.     

The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA.

Low salt extracts of chicken oviduct nuclei contain a DNA binding protein with high affinity for specific DNA sequences in the flanking regions of the chicken lysozyme gene. Two of the three binding sites found within a total of 11 kb upstream from the promoter are located only 92 bp apart from each other. Upon comparison of the DNA binding sites, the symmetrical consensus sequence 5'- TGGCANNNTGCCA -3' can be deduced as the protein recognition site. This sequence is the central part of 23 to 25 base pairs protected by the DNA binding protein from DNAase I digestion. A homologous binding activity can be detected in nuclei from several chicken tissues and from mouse liver.     

Hoechst 33258, distamycin A,and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.by E.R. Schmidt     

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.     

Specific interaction of mouse major satellite with MAR-binding protein SAF-A

A DNA-binding activity specific to the major mouse satellite (satMa) has been detected in a nuclear matrix protein extract by electrophoretic mobility shift assays (EMSA) after fractionation by ion exchange chromatography. An antibody raised against the satMa-protein complexes recovered from preparative EMSA recognizes on Western blots one major polypeptide with an apparent molecular mass of 120 kDa. The protein also has a similar affinity for a matrix-associated region (MAR) fragment. We demonstrate that the protein is a murine homologue of SAF-A which has been shown to bind selectively to MARs and is responsible for the satMa-binding activity in the chromatographic fractions. SatMa has significant homology to the mouse minor satellite fragments, but its binding of SAF-A shows much less affinity. No protected regions of significant length were found by footprinting, but multiple T residues scattered within the satMa sequence are protected, indicating that the whole fragment is involved in the binding to SAF-A. Combined immunofluorescence (SAF-A) and FISH (satMa) with in situ nuclear matrix procedures reveal that SAF-A and satMa colocalize. SAF-A appears as bright dots in interphase nuclei, presumably associated with MARs, predominantly surrounding and covering heterochromatic areas. A scheme based on morphological observations and biochemical data of SAF-A double satMa/MAR specificity is discussed.