Showing papers on "Histone binding published in 2009"

H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies.

Abstract: Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.

Distinctive sequence patterns in metazoan and yeast nucleosomes: Implications for linker histone binding to AT-rich and methylated DNA

Abstract: Linker histones (LHs) bind to the DNA entry/exit points of nucleosomes and demonstrate preference for AT-rich DNA, although the recognized sequence patterns remain unknown. These patterns are expected to be more pronounced in metazoan nucleosomes with abundant LHs, compared to yeast nucleosomes with few LHs. To test this hypothesis, we compared the nucleosome core particle (NCP) sequences from chicken, Drosophila and yeast, extending them by the flanking sequences extracted from the genomes. We found that the known approximately 10-bp periodic oscillation of AT-rich elements goes beyond the ends of yeast nucleosomes, but is distorted in metazoan sequences where the 'out-of-phase' AT-peaks appear at the NCP ends. The observed difference is likely to be associated with sequence-specific LH binding. We therefore propose a new structural model for LH binding to metazoan nucleosomes, postulating that the highly conserved nonpolar 'wing' region of the LH globular domain (tetrapeptide GVGA) recognizes AT-rich fragments through hydrophobic interactions with the thymine methyl groups. These interactions lead to DNA bending at the NCP ends and formation of a 'stem-like' structure. The same mechanism accounts for the high affinity of LH to methylated DNA-a feature critical for stabilization of the higher-order structure of chromatin and for repression of transcription.

Alternative splicing of NURF301 generates distinct NURF chromatin remodeling complexes with altered modified histone binding specificities.

Abstract: Drosophila NURF is an ISWI–containing chromatin remodeling complex that catalyzes ATP–dependent nucleosome sliding. By sliding nucleosomes, NURF can alter chromatin structure and regulate transcription. NURF301/BPTF is the only NURF–specific subunit of NURF and is instrumental in recruiting the complex to target genes. Here we demonstrate that three NURF301 isoforms are expressed and that these encode functionally distinct NURF chromatin remodeling complexes. Full-length NURF301 contains a C-terminal bromodomain and juxtaposed PHD finger that bind histone H3 trimethylated at Lys4 (H3K4me3) and histone H4 acetylated at Lys16 (H4K16Ac) respectively. However, a NURF301 isoform that lacks these C-terminal domains is also detected. This truncated NURF301 isoform assembles a complex containing ISWI, NURF55, and NURF38, indicating that a second class of NURF remodeling complex, deficient in H3K4me3 and H4K16Ac recognition, exists. By comparing microarray expression profiles and phenotypes of null Nurf301 mutants with mutants that remove the C-terminal PHD fingers and bromodomain, we show that full-length NURF301 is not essential for correct expression of the majority of NURF gene targets in larvae. However, full-length NURF301 is required for spermatogenesis. Mutants that lack full-length NURF exhibit a spermatocyte arrest phenotype and fail to express a subset of spermatid differentiation genes. Our data reveal that variants of the NURF ATP–dependent chromatin remodeling complex that recognize post-translational histone modifications are important regulators of primary spermatocyte differentiation in Drosophila.

Linker Histones Stimulate HSPA2 ATPase Activity Through NASP Binding and Inhibit CDC2/Cyclin B1 Complex Formation During Meiosis in the Mouse

Abstract: In mammalian spermatocytes, cell division cycle protein 2 (CDC2)/cyclin B1 and the chaperone heat shock protein A2 (HSPA2) are required for the G2→M transition in prophase I. Here, we demonstrate that in primary spermatocytes, linker histone chaperone testis/embryo form of nuclear autoantigenic sperm protein (tNASP) binds the heat shock protein HSPA2, which localizes on the synaptonemal complex of spermatocytes. Significantly, the tNASP-HSPA2 complex binds linker histones and CDC2, forming a larger complex. We demonstrate that increasing amounts of tNASP favor tNASP-HSPA2-CDC2 complex formation. Binding of linker histones to tNASP significantly increases HSPA2 ATPase activity and the capacity of tNASP to bind HSPA2 and CDC2, precluding CDC2/cyclin B1 complex formation and, consequently, decreasing CDC2/cyclin B1 kinase activity. Linker histone binding to NASP controls the ability of HSPA2 to activate CDC2 for CDC2/cyclin B1 complex formation; therefore, tNASP's role is to provide the functional link between linker histones and cell cycle progression during meiosis.

Epigenetic targets in drug discovery

Abstract: PART I: General Aspects and Methodologies NEW FRONTIERS IN EPIGENETIC MODIFICATIONS Introduction DNA Methylation Histone Modifications and the Histone Code Hypothesis Origins of Specificity in Histone Binding Proteins/Modifying Enzymes Histone Modification Cross-Talk Inhibitors of DNMTs and HDACs STRUCTURAL BIOLOGY OF EPIGENETIC TARGETS Introduction Histone Acetyltransferases Histone Deacetylases Sirtuins Histone Methylation Enzymes Histone Demethylases DNA Methyltransferases Concluding Remarks: The Challenge of Structural Studies of Epigenetic Targets COMPUTER- AND STRUCTURE-BASED LEAD IDENTIFICATION FOR EPIGENETIC TARGETS Introduction Computer-Based Methods in Drug Discovery DNA Methyltransferases Histone Deacetylases Histone Acetyltransferases Histone Methyltransferases Histone Demethylases HISTONE MODIFICATION ANALYSIS USING MASS SPECTROMETRY Introduction Histone Molecules Capillary Electrophoresis Reversed Phase Chromatography Mass Spectrometry IN VITRO ASSAYS FOR HISTONE-MODIFYING ENZYMES Introduction Screening Hierarchy General Principles of Screening for Histone-Modifying Enzymes Histone Deacetylases Histone Acetyltransferases Histone Methyltransferases Histone Demethylases EPIGENETIC TARGETS IN DRUG DISCOVERY: CELL-BASED ASSAYS FOR HDAC INHIBITOR HIT VALIDATION Introduction In Vitro Assays for HDAC Inhibitors Cell-Based Assays Biological Function-Based Assays HDAC Isoform Functional Assays Anticancer Activity Perspective CHROMATIN IMMUNOPRECIPITATION CHIP: WET LAB MEETS IN SILICO Background and Overview The Basic Principle of ChIP Different Variations of ChIP Analysis of ChIP Data Data Analysis Examples The Future of ChIP PART II: Epigenetic Target Classes and Inhibitor Development DNA METHYLTRANSFERASE INHIBITORS Introduction DNA Methylation DNA Methyltransferases Biochemical Mechanism of DNA Methylation Physiological Role of DNA Methylation DNA Methylation and Disease DNMT Inhibitors Therapeutic Applications of DNMT Inhibitors HISTONE DEACETYLASE INHIBITORS Introduction Mechanism and X-Ray Crystal Structure Histone Deacetylase Inhibitors NAD-DEPENDENT DEACETYLASES AS THERAPEUTIC TARGETS Historical Perspective and Functions of Sir2 in Yeast Sirtuin Enzymatic Activity and its Modulation by Endogenous Molecules Small Molecule Inhibitors of Sirtuins Small Molecule Activators of Sirtuins SIRT1 as a Therapeutic Target in Metabolic Syndrome and Type 2 Diabetes Neurological Diseases INHIBITORS OF HISTONE ACETYLTRANSFERASES: DISCOVERY AND BIOMEDICAL PERSPECTIVES Introduction HAT Inhibitors HISTONE METHYLTRANSFERASES AS NOVEL DRUG TARGETS Introduction Protein Methylation Histone Demethylation Histone Methyltransferases Histone Methyltransferase Inhibitors HISTONE DEMETHYLASES Introduction Chromatin Histone Methylation and Demethylation Histone Demethylases LSD1/KDM1 JmjC Domain-Containing Demethylases

Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

Abstract: Background The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays.

Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis

Abstract: Increasing evidence indicates that an important consequence of protein posttranslational modification (PTM) is the creation of a high affinity binding site for the selective interaction with a PTM-specific binding protein (BP). This PTM-mediated interaction is typically required for downstream signaling propagation and corresponding biological responses. Because the vast majority of mammalian proteins contain PTMs, there is an immediate need to discover and characterize previously undescribed PTMBPs. To this end, we developed and validated an innovative in vivo approach called mammalian tethered catalysis (MTeC). By using methylated histones and methyl-specific histone binding proteins as the proof-of-principle, we determined that the new MTeC approach can compliment existing in vitro binding methods, and can also provide unique in vivo insights into PTM-dependent interactions. For example, we confirmed previous in vitro findings that endogenous HP1 preferentially binds H3K9me3. However, in contrast to recent in vitro observations, MTeC revealed that the tandem tudor domain-containing proteins, JMJD2A and 53BP1, display no preferential H4K20 methyl-selectivity in vivo. Last, by using MTeC in an unbiased manner to identify H3K9 methyl-specific PTMBPs, we determined that endogenous G9a binds methylated H3K9 in vivo. Further use of MTeC to characterize this interaction revealed that G9a selectively binds H3K9me1 in vivo, but not H3K9me2, contrary to recent in vitro findings. Although this study focused solely on methylated histones, we demonstrate how the innovative MTeC approach could be used to identify and characterize any PTMBP that binds any PTM on any protein in vivo.

Histones induce the release of apoptogenic factors from liver mitochondria

Abstract: The effect of histones on the release of apoptogenic factors has been studied. The incubation of H1 histone or total histones with mitochondria from a rat liver results in their binding to mitochondria. Furthermore, histones induce the release of cytochrome c and a number of other proteins from the intermembranous space of mitochondria. Proteins released from mitochondria in the presence of histones exhibit apoptogenic activity and induce internucleosomal DNA fragmentation of thymus nuclei. The cytotoxic effect of histones is probably mediated by apoptogenic proteins, which are released from intermembranous space as a response of histone binding to mitochondria.

Highly Preferential Linker Histone Binding to Actinomycin D-Treated DNA

Abstract: Electromobility-shift assay (EMSA) was applied for studying the competition between the anticancer antibiotic actinomycin D (ACTD) and linker histones H1 and H5 for binding to DNA. ACTD is widely used in the treatment of some malignancies. Its binding to DNA occurs in two steps: initial intercalation between guanine and cytosine bases followed by invading the minor groove of the polynucleotide chain. As a consequence of this structural perturbation of DNA, a strong interference with the binding of important regulatory and functional proteins to DNA may occur. Here we present our recent experimental data obtained in a drug-competition assay between ACTD and linker histones H1 and H5 under the following conditions: i) simultaneous incubation of DNA with the antibiotic and histone; ii) preincubation of DNA with ACTD and subsequent addition of the histone; and iii) preincubation of DNA with histone and subsequent addition of the drug. Surprisingly, linker histones bound more effectively to ACTD-pretre...