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-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.The N-terminal tails of histones are subject to many reversible covalent modifications in vivo, and different modifications have often been associated with either active or repressed chromatin states. According to prevailing ideas, the status of the cell is translated to chromatin in the form of specific post-translational modification (PTM) 4 patterns on histone tails. This tagged chromatin is then recognized by effector proteins and complexes that regulate how the underlying genetic information is used (1). The complicated and intertwined processes of tagging the histone tails, recognizing the tags, remodeling chromatin into active (open) or repressed (compacted) states, and removing the tags requires the coordination of multiple protein functions.The nucleosome remodeling and histone deacetylase (NuRD) complex is unique among nucleosome remodeling complexes in that it couples histone deacetylase activity with nucleosome remodeling ATPase activity (although the purpose of this enzymatic combination is currently unclear). The NuRD complex has traditionally been considered a transcriptional corepressor complex, consistent with the repressive function of histone deacetylation (reviewed in Refs. 2, 3). Several key NuRD complex components have been shown to play a role in development and cell lineage commitment in multiple contexts. For example, in Caenorhabditis elegans, the CHD4 homologue let-418 is required for the repression of germ line ...

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis for their association with chromatin. We found that PHD1/2 targets nucleosomes in a multivalent manner, concomitantly engaging two histone H3 tails. This robust synergistic interaction displaces HP1γ from pericentric sites, inducing changes in chromatin structure and leading to the dispersion of the heterochromatic mark H3K9me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

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CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3,Thr6 or Ser10 abolishes this interaction. A model of the PHD2–H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA (Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5 splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine ''ladder.'' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.protein structure ͉ RanBP2 zinc fingers ͉ RNA-binding proteins ͉ splicing A lmost all human genes are thought to be alternatively spliced, and it has been estimated that at least 15% of human diseases are associated with changes in RNA processing (1). The selection of splice sites is influenced heavily by the binding of accessory splicing factors to regulatory sequences in the pre-mRNA. SR proteins are splicing factors that contain a C-terminal Arg/Ser-rich (RS) domain and either 1 or 2 N-terminal RNA recognition motifs (RRMs) (2). They play crucial roles in constitutive and alternative splicing, promoting recognition of splice sites by binding to exonic splicing enhancers (ESEs). RRM domains bind ssRNA with high affinity in a sequence-specific manner, whereas RS domains appear to facilitate both protein-protein and protein-RNA interactions (3, 4). Other RS domain-containing proteins that lack a canonical RRM, termed ''SR-like'' proteins (see, for example, ref. 5) are also known to play roles in splicing.ZRANB2 (Zis, ZNF265) is an SR-like nuclear protein that is expressed in most tissues and is conserved between nematodes and mammals. It interacts with the spliceosomal proteins U1-70K and U2AF 35 and can alter the distribution of splice variants of GluR-B, SMN2, and Tra2␤ in minigene reporter assays (6, 7). As such, ZRANB2 appears to regulate splice site choice. However, in place of the canonical RNA-binding RRM domains, ZRANB2 displays 2 N-terminal RanBP2-type zinc fingers (ZnFs).RanBP2-type ZnF domains are defined by the consensus sequence W-X-C-X 2-4 -C-X 3 -N-X 6 -C-X 2 -C. They occur multiple times in at least 21 human proteins, and the fold comprises 2 distorted ␤-hairpins sandwiching a central tryptophan residue and ...