Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 A resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.

Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases, members of a newly emerging transcription-factor family, remove methyl groups from the lysine residues of the histone tails and thereby regulate the transcriptional activity of target genes. JmjC-domain-containing proteins have been predicted to be demethylases. For example, the JmjC-containing protein JMJD2A has been characterized as a H3-K9me3- and H3-K36me3-specific demethylase. Here, structures of the catalytic-core domain of JMJD2A with and without alpha-ketoglutarate in the presence of Fe2+ have been determined by X-ray crystallography. The structure of the core domain, consisting of the JmjN domain, the JmjC domain, the C-terminal domain, and a zinc-finger motif, revealed the unique elements that form a potential substrate binding pocket. Sited-directed mutagenesis in conjunction with demethylase activity assays allowed us to propose a molecular model for substrate selection by the JMJD2 histone demethylase family.

The PHD finger motif is a signature chromatin-associated motif that is found throughout eukaryotic proteomes. Here we have determined the histone methyl-lysine binding activity of the PHD fingers present within the Saccharomyces cerevisiae proteome. We provide evidence on the genomic scale that PHD fingers constitute a general class of effector modules for histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 trimethylated at lysine 36 (H3K36me3). Structural modeling of PHD fingers demonstrates a conserved mechanism for recognizing the trimethyl moiety and provides insight into the molecular basis of affinity for the different methyl-histone ligands. Together, our study suggests that a common function for PHD fingers is to transduce methyl-lysine events and sheds light on how a single histone modification can be linked to multiple biological outcomes.

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 (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.

Anopheles gambiae mosquitoes that transmit Plasmodium falciparum malaria use a series of olfactory cues present in human sweat to locate their hosts for a blood meal. Recognition of these odor cues occurs through the interplay of odorant receptors and odorant-binding proteins (OBPs) that bind to odorant molecules and transport and present them to the receptors. Recent studies have implicated potential heterodimeric interactions between two OBPs, OBP1 and OBP4, as important for perception of indole by the mosquito (Biessmann, H., Andronopoulou, E., Biessmann, M. R., Douris, V., Dimitratos, S. D., Eliopoulos, E., Guerin, P. M., Iatrou, K., Justice, R. W., Kröber, T., Marinotti, O., Tsitoura, P., Woods, D. F., and Walter, M. F. (2010) PLoS ONE 5, e9471; Qiao, H., He, X., Schymura, D., Ban, L., Field, L., Dani, F. R., Michelucci, E., Caputo, B., della Torre, A., Iatrou, K., Zhou, J. J., Krieger, J., and Pelosi, P. (2011) Cell. Mol. Life Sci. 68, 1799 -1813). Here we present the 2.0 Å crystal structure of the OBP4-indole complex, which adopts a classical odorant-binding protein fold, with indole bound at one end of a central hydrophobic cavity. Solution-based NMR studies reveal that OBP4 exists in a molten globule state and binding of indole induces a dramatic conformational shift to a well ordered structure, and this leads to the formation of the binding site for OBP1. Analysis of the OBP4-OBP1 interaction reveals a network of contacts between residues in the OBP1 binding site and the core of the protein and suggests how the interaction of the two proteins can alter the binding affinity for ligands. These studies provide evidence that conformational ordering plays a key role in regulating heteromeric interactions between OBPs.Anopheles gambiae mosquitoes are the primary vectors for malaria caused by Plasmodium falciparum and have an extremely high preference for feeding on human hosts (1, 2) and are attracted to odor molecules from incubated human sweat and other skin emanations (3-6). Disrupting the normal olfactory responses to these odors presents an attractive tool to combat transmission of malaria and other mosquito borne diseases, and a number of efforts are now under way to discover novel reagents for this purpose.In insects the detection of odorants occurs primarily in the olfactory sensilla and involves the interplay of membranebound olfactory receptors (7) and odorant-binding proteins (OBPs), 3 which are expressed into the lymph fluid that surrounds the olfactory dendrites (8) where they can reach concentrations in the millimolar range (9, 10). OBPs have multiple roles including protecting odors from degradation and transporting them to the olfactory receptors (11, 12). There is evidence that OBPs have two primary roles in odorant perception. In the first model a number of groups have proposed that OBPs act as passive carriers for the odorant, and pH changes in the vicinity of the dendritic membrane lead to conformational changes that stimulate ligand release, freeing the ligand to activate the receptor (1...

The interaction of PtdIns(5)P with the tumor suppressor protein ING2 has been implicated in the regulation of chromatin modification. To enhance the stability of PtdIns(5)P for studies of the biological role in vivo, two phosphatase-resistant moieties were used to replace the labile 5-phosphate. The total asymmetric synthesis of the 5-methylenephosphonate (MP) and 5-phosphothionate (PT) analogues of PtdIns(5)P is described herein, and the resulting metabolically stabilized lipid analogues were evaluated in three ways. First, liposomes containing either the dioleoyl MP or PT analogues bound to recombinant ING2 similar to liposomes containing dipalmitoyl PtdIns(5)P, indicating that the replacement of the hydrolyzable 5-phosphate group does not compromise the binding. Second, the dioleoyl MP and PT PtdIns(5)P analogues were equivalent to dipalmitoyl PtdIns(5)P in augmenting cell death induced by a DNA double-strand break in HT1080 cells. Finally, molecular modeling and docking of the MP or PT analogues to the C-terminus PtdInsP-binding region of ING2 (consisting of a PHD finger and a polybasic region) revealed a number of complementary surface and electrostatic contacts between the lipids and ING2.