MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties

The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.     

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.     

The Drosophila methyl-DNA binding protein MBD2/3 interacts with the NuRD complex via p55 and MI-2

Background  

Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications. MBD2/3 is the only candidate gene in the Drosophila genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the Nucleosome Remodelling and Deacetylase (NuRD) complex, respectively. An association of Drosophila MBD2/3 with the Drosophila NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.     

p66alpha and p66beta of the Mi-2/NuRD complex mediate MBD2 and histone interaction

The Mi-2/NuRD complex is a multi-subunit protein complex with enzymatic activities involving chromatin remodeling and histone deacetylation. Targeting of Mi-2/NuRD to methylated CpG sequences mediates gene repression. The function of p66α and of p66β within the multiple subunits has not been addressed. Here, we analyzed the in vivo function and binding of both p66-paralogs. Both factors function in synergy, since knocking-down p66α affects the repressive function of p66β and vice versa. Both proteins interact with MBD2 functionally and biochemically. Mutation of a single amino acid of p66α abolishes in vivo binding to MBD2 and interferes with MBD2-mediated repression. This loss of binding results in a diffuse nuclear localization in contrast to wild-type p66α that shows a speckled nuclear distribution. Furthermore, wild-type subnuclear distribution of p66α and p66β depends on the presence of MBD2. Both proteins interact with the tails of all octamer histones in vitro, and acetylation of histone tails interferes with p66 binding. The conserved region 2 of p66α is required for histone tail interaction as well as for wild-type subnuclear distribution. These results suggest a two-interaction forward feedback binding mode, with a stable chromatin association only after deacetylation of the histones has occurred.     

Molecular association between ATR and two components of the nucleosome remodeling and deacetylating complex, HDAC2 and CHD4.

Ataxia telangiectasia mutated (ATM)- and Rad3-related protein (ATR) is a phosphatidylinositol-kinase (PIK)-related kinase that has been implicated in the response of human cells to multiple forms of DNA damage and may play a role in the DNA replication checkpoint. The purification of an ATR complex allowed identification of chromodomain-helicase-DNA-binding protein 4 (CHD4) as an ATR-associated protein by tandem mass spectrometric sequencing. CHD4 (also called Mi-2beta) is a component of a histone-deacetylase-2 (HDAC2)-containing complex, the nucleosome remodeling and deacetylating (NRD) complex. Endogenous ATR, CHD4, and HDAC2 are shown to coimmunoprecipitate, and ATR and HDAC2 coelute through two biochemical purification steps. Other members of the NRD complex, HDAC1, MTA1, and MTA2, are also detectable in ATR immunoprecipitates. ATR's association with CHD4 and HDAC2 suggests that there may be a linkage between ATR's role in mediating checkpoints induced by DNA damage and chromatin modulation via remodeling and deacetylation.     

AlphaB-crystallin phosphorylated at Ser-59 is localized in centrosomes and midbodies during mitosis

We have reported that the three serine residues in alphaB-crystallin are phosphorylated under various stress conditions. We prepared affinity-purified antibodies recognizing each of the phosphorylated serine residues (Ser-19, Ser-45, and Ser-59, respectively) in alphaB-crystallin with peptides (p19S, p45S, or p59S) that contained the corresponding phosphorylated serine residue. Immunocytochemically anti-p45S antibodies stained the cytoplasm of mitotic cells (J. Biol. Chem. 273, 28,346-28,354). We have now found that the anti-p59S antibodies recognize centrosomes and midbodies of dividing cells. alphaB-Crystallin was the only protein recognized by the anti-p59S antibodies in Western blot analyses of isolated centrosome fractions. alphaB-Crystallin phosphorylated at Ser-59 was localized at the microtubule organizing centers by means of double staining with anti-beta-tubulin antibody in aster formation analysis and was co-localized with gamma-tubulin in centrosomes. Gamma-Tubulin was co-immunoprecipitated with alphaB-crystallin in U373 glioma cell extracts. On the other hand, the location of the phosphorylated alphaB-crystallin deviated from that of alpha-tubulin or gamma-tubulin in the midbody region. Taken together with the evidences that several chaperones are distributed to centrosomes, these results suggest that alphaB-crystallin as a chaperone might be also involved in the quality control of proteins.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.     

Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1

Corepressors N-CoR and SMRT participate in diverse repression pathways and exist in large protein complexes including HDAC3, TBL1 and TBLR1. However, the roles of these proteins in SMRT-N-CoR complex function are largely unknown. Here we report the purification and functional characterization of the human N-CoR complex. The purified N-CoR complex contains 10-12 associated proteins, including previously identified components and a novel actin-binding protein IR10. We show that TBL1/TBLR1 associates with N-CoR through two independent interactions: the N-terminal region and the C-terminal WD-40 repeats interact with the N-CoR RD1 and RD4 region, respectively. In vitro, TBL1/TBLR1 bind histones H2B and H4, and, importantly, repression by TBL1/TBLR1 correlates with their interaction with histones. By using specific small interference RNAs (siRNAs), we demonstrate that HDAC3 is essential, whereas TBL1 and TBLR1 are functionally redundant but essential for repression by unliganded thyroid hormone receptor. Together, our data reveal the roles of HDAC3 and TBL/TBLR1 and provide evidence for the functional importance of histone interaction in repression mediated by SMRT-N-CoR complexes.     

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses.

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis

Circadian timing is generated through a unique series of autoregulatory interactions termed the molecular clock. Behavioral rhythms subject to the molecular clock are well characterized. We demonstrate a role for Bmal1 and Clock in the regulation of glucose homeostasis. Inactivation of the known clock components Bmal1 (Mop3) and Clock suppress the diurnal variation in glucose and triglycerides. Gluconeogenesis is abolished by deletion of Bmal1 and is depressed in Clock mutants, but the counterregulatory response of corticosterone and glucagon to insulin-induced hypoglycaemia is retained. Furthermore, a high-fat diet modulates carbohydrate metabolism by amplifying circadian variation in glucose tolerance and insulin sensitivity, and mutation of Clock restores the chow-fed phenotype. Bmal1 and Clock, genes that function in the core molecular clock, exert profound control over recovery from insulin-induced hypoglycaemia. Furthermore, asynchronous dietary cues may modify glucose homeostasis via their interactions with peripheral molecular clocks.     

The two functions of herpes simplex virus 1 ICP0, inhibition of silencing by the CoREST/REST/HDAC complex and degradation of PML, are executed in tandem

ICP0, an α (immediate-early) protein of herpes simplex virus 1, performs at least two key functions. It blocks inhibition of viral-gene expression by interferon, a function dependent on the degradation of the ND10 components PML and SP100 by the ubiquitin ligase expressed by the RING finger (RF), and it blocks silencing of viral DNA mediated by the HDAC1/2-CoREST-REST complex. In the latter case, a mutant CoREST lacking the HDAC1 binding site compensates totally or in part for the absence of ICP0 in a cell-type-dependent manner. Here, we compare the phenotypes of an ICP0 mutant containing disabling amino acid substitutions in the RF with those of a mutant with substitutions in the CoREST binding site (R8507). We report the following: (i) the onset of replication of both mutants was delayed, but the RF mutant yields did not reach wild-type virus levels even as late as 48 h after infection, and (ii) in infected cells, PML is rapidly degraded by wild-type virus, with some delay by the R8507 mutant, and is spared by the RF mutant. The translocation of ICP0 to the cytoplasm is impaired in cells infected with the RF mutant or delayed in cells infected with the R8507 mutant. Finally, in contrast to wild-type viruses, both mutants are inhibited by alpha or gamma interferon. The results indicate that both sets of events, the degradation of PML and the blocking of silencing, are interdependent and in large measure dependent on events in the ND10 nuclear bodies.