dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines.     

The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization

Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity.     

dMi-2 chromatin binding and remodeling activities are regulated by dCK2 phosphorylation

A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.     

PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor

PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.     

The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells

Chromosomal replication results in the duplication not only of DNA sequence but also of the patterns of histone modification, DNA methylation, and nucleoprotein structure that constitute epigenetic information. Pericentromeric heterochromatin in human cells is characterized by unique patterns of histone and DNA modification. Here, we describe association of the Mi-2/NuRD complex with specific segments of pericentromeric heterochromatin consisting of Satellite II/III DNA located on human chromosomes 1, 9, and 16 in some but not all cell types. This association is linked in part to DNA replication and chromatin assembly and may suggest a role in these processes. Mi-2/NuRD accumulation is independent of Polycomb association and is characterized by a unique pattern of histone modification. We propose that Mi-2/NuRD constitutes an enzymatic component of a pathway for assembly and maturation of chromatin utilized by rapidly proliferating lymphoid cells for replication of constitutive heterochromatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant homeodomain (PHD) fingers of CHD4 are histone H3-binding modules with preference for unmodified H3K4 and methylated H3K9

A major challenge in chromatin biology is to understand the mechanisms by which chromatin is remodeled into active or inactive states as required during development and cell differentiation. One complex implicated in these processes is the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains both histone deacetylase and nucleosome remodeling activities and has been implicated in the silencing of subsets of genes involved in various stages of cellular development. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core component of the NuRD complex and contains a nucleosome remodeling ATPase domain along with two chromodomains and two plant homeodomain (PHD) fingers. We have previously demonstrated that the second PHD finger of CHD4 binds peptides corresponding to the N terminus of histone H3 methylated at Lys(9). Here, we determine the solution structure of PHD2 in complex with H3K9me3, revealing the molecular basis of histone recognition, including a cation-π recognition mechanism for methylated Lys(9). Additionally, we demonstrate that the first PHD finger also exhibits binding to the N terminus of H3, and we establish the histone-binding surface of this domain. This is the first instance where histone binding ability has been demonstrated for two separate PHD modules within the one protein. These findings suggest that CHD4 could bind to two H3 N-terminal tails on the same nucleosome or on two separate nucleosomes simultaneously, presenting exciting implications for the mechanism by which CHD4 and the NuRD complex could direct chromatin remodeling.     

Different Mi-2 complexes for various developmental functions in Caenorhabditis elegans

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.