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Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair (1-6). Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module (7-10) embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization., The essential heterodimeric chaperone FACT destabilizes nucleosomes to promote polymerase progression on chromatin templates (1-4,11) and maintains chromatin structure in vivo (5,6). The recognition of the histone H2A-H2B heterodimer is [...]

A study is conducted to characterize the mechanism and scope of lysine-specific demethylase 1 (LSD1) inhibition by a propargylamine-derivatized histone H3 substrate. In this mechanism, oxidation of the amine yields the propargylic iminium ion.

The synthesis of trifluoromethyl phenyl benzamides and their effect on histone acetyltransferase (HAT) activity of p300 is reported. It is found that pentadecyl hydrocarbon chain of (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide) is required to activate the HAT under certain context.

The inhibition of lysine-specific demethylase 1 (LSD1) by monoamine oxidase (MAOs) through kinetic analysis and in a more quantitative manner is investigated. It is shown that 2-phenylcyclopropylamine (2-PCPA) is a time-dependent, mechanism-based irreversible inhibitor of LSD1 and a covalent intermediate is formed between 2-PCPA and the noncovalent FAD cofactor of LSD1.

Ttwo stereochemical variants of the natural product are designed and synthesized. Results reveals the identity of the recognition pattern between azumamides.

Glutamine-rich sequences exist in a wide range of proteins across multiple species. A subset of glutamine-rich sequences has been shown to form amyloid fibers implicated in human diseases. The physiological functions of these sequence motifs are not well understood, partly because of the lack of structural information. Here we have determined a high-resolution structure of a glutamine-rich domain from human histone deacetylase 4 (HDAC4) by x-ray crystallography. The glutamine-rich domain of HDAC4 (19 glutamines of 68 residues) folds into a straight [alpha]-helix that assembles as a tetramer. In contrast to most coiled coil proteins, the HDAC4 tetramer lacks regularly arranged apolar residues and an extended hydrophobic core. Instead, the protein interfaces consist of multiple hydrophobic patches interspersed with polar interaction networks, wherein clusters of glutamines engage in extensive intra-and interhelical interactions. In solution, the HDAC4 tetramer undergoes rapid equilibrium with monomer and intermediate species. Structure-guided mutations that expand or disrupt hydrophobic patches drive the equilibrium toward the tetramer or monomer, respectively. We propose that a general role of glutamine-rich motifs be to mediate protein-protein interactions characteristic of a large component of polar interaction networks that may facilitate reversible assembly and disassembly of protein complexes. amyloid | transcription

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-I[beta]/INHAT at a resolution of 2.3 [Angstrom]. We found that SET/TAF-I[beta]/INHAT formed a dimer that assumed a 'headphone'-like structure. Each subunit of the SET/ TAF-I[beta]/INHAT dimer consisted of an N terminus, a backbone helix, and an 'earmuff' domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-I[beta]/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by [approximately equal to]40[degrees]. Our biochemical analyses of mutants revealed that the region of SET/TAF-I[beta]/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-I[beta]/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures. nucleosome | chromatin | transcription | replication | repair

The silent information regulator 2 (Sir2) family of proteins (sirtuins or SIRTs), which belong to class III histone/protein deacetylases, have been implicated in calorie restriction, aging, and inflammation. We hypothesized that cigarette smoke-mediated proinflammatory cytokine release is regulated by SIRT1 by its interaction with NF-[kappa]B in a monocyte-macrophage cell line (MonoMac6) and in inflammatory cells of rat lungs. Cigarette smoke extract (CSE) exposure to MonoMac6 cells caused dose- and time-dependent decreases in SIRT1 activity and levels, which was concomitant to increased NF-[kappa]B-dependent proinflammatory mediator release. Similar decrements in SIRT1 were also observed in inflammatory cells in the lungs of rats exposed to cigarette smoke as well as with increased levels of several NF-[kappa]B-dependent proinflammatory mediators in bronchoalveolar lavage fluid and in lungs. Sirtinol, an inhibitor of SIRT1, augmented, whereas resveratrol, an activator of SIRT1, inhibited CSE-mediated proinflammatory cytokine release. CSE-mediated inhibition of SIRT1 was associated with increased NF-[kappa]B levels. Furthermore, we showed that SIRT1 interacts with the RelA/p65 subunit of NF-[kappa]B, which was disrupted by cigarette smoke, leading to increased acetylation RelA/ p65 in MonoMac6 cells. Thus our data show that SIRT1 regulates cigarette smoke-mediated proinflammatory mediator release via NF[kappa]B, implicating a role of SIRT1 in sustained inflammation and aging of the lungs. oxidants; resveratrol; MonoMac6 cells; chronic obstructive pulmonary disease; histone; acetylation; deacetylase

A study finds out how HiNF-P is regulated during the cell-cycle and examines its stability relative to [p220.sup.NPAT] using serum-stimulated cells and cells synchronized in S-phase and in mitosis. It was observed that while HiNF-P is maintained at steady-state levels in the complete cell cycle, the proteasome pathway actively degrades both HiNF-P and [p220.sup.NPAT].

The molecular basis for variation in vernalization in Arabidopsis natural accessions adapted to different climates is investigated. The results have shown that the variation in epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC) have contributed to Arabidopsis adaptation.

An approach to follow new histone deposition upon ultraviolet (UV) irradiation in human cells is devised. It is shown that new H3.1 histones get incorporated in vivo at repair sites and it is concluded that chromatin restoration after damage cannot rely simply on histone recycling.

The crystal structure of anti-silencing function 1 (Asf1) bound to histones H3/H4 is determined, which reveals how a histone chaperone binds histones and how the structures of H3 and H4 differ outside of the histone octamer. The Asf1-H3/H4 structure has indicated a 'strand-capture' mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that might be used by histone chaperones.

An attempt is made to reconcile some of the inconsistencies identified in a collection of proteins to selectively recognize di-and trimethyl H3K4, marks that are associated with actively transcribed genes. It is revealed that factors known to be involved with active transcription associate with H3K4me2/3 has important significance for understanding the functional role of this histone modification.

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1 [beta]-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxiatelangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression. Cajal bodies | coiled bodies | cell cycle

Lysine-specific demethylase 1 (LSD1) was recently identified as the first histone demethylase that specifically demethylates monomethylated and dimethylated histone H3 at K4. It is a component of the CoREST and other corepressor complexes and plays an important role in silencing neuronal-specific genes in nonneuronal cells, but the molecular mechanisms of its action remain unclear. The 2.8-[Angstrom]-resolution crystal structure of the human LSD1 reveals that LSD1 defines a new subfamily of FAD-dependent oxidases. The active center of LSD1 is characterized by a remarkable 1,245-[[Angstrom].sup.3] substrate-binding cavity with a highly negative electrostatic potential. Although the protein core of LSD1 resembles other flavoenzymes, its enzymatic activity and functions require two additional structural modules: an N-terminal SWIRM domain important for protein stability and a large insertion in the catalytic domain indispensable both for the demethylase activity and the interaction with CoREST. These results provide a framework for further probing the catalytic mechanism and the functional roles of LSD1. histonemodification | flavoenzyme | catalysis

Fluorescence energy transfer and scanning force microscopy were used to explore the influence of the salt concentration, linker histon H1, and histone acetylation on the structure of trinucleosomes reconstituted on a 608 bp DNA containing one centered nucleosome positioning signal. The effect of acetylation of all histones is found to lead to decomposition, measured as an increased distance between the DNA ends, and also increased the internucleosomal distances.

A novel flowering gene that is under polycomb-group (PcG) control in Arabidopis-the MADS-box gene AGL19 is identified. Epistasis analyses revealed that AGL19 works in the poorly characterized FLC-independent vernalization pathway and does not require SOC1 to function.

Three natural and two synthetic sites are compared to determine the relative strength of different histone octamer-binding sites. It is inferred that sequence selection by the histone octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters contribute differently to nucleosome formation.

Isothermal titration calorimetry (ITC) is used to analyze the thermodynamics of the interactions of importin [alpha] with [beta], [alpha] with pentameric histone chaperone nucleoplasmin (NP), and the [alpha]/[beta] heterodimer with NP. The data obtained revealed that binding of both importin [alpha] and [alpha]/[beta] to NP is mediated by a favorable enthalpic contribution and that NP could accommodate up to five importin molecules per NP pentamer.

The article presents a comparative analysis of the ability of the polycomb group (PcG) proteins to bind and to inhibit the remodeling of the numerous nucleosomal templates. The posterior sex combs are shown to easily bind to the mononucleosome templates, while exhibiting a poor remodeling.

The design and synthesis of stable and nonisomerizable phosphohistidine (pHis) analogues is described. The pHis analogues are used in solid-phase peptide synthesis and semi-synthesis of histone H4 and the first antibody that recognizes pHis is obtained by using the synthetic peptide as the immunogen.

The studies have shown that in poly(ADP-ribose) glycohydrolase (PARG) null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A and H2B. It is shown that greater DNA accessibility, increased DNA damage, and increased cell death have contributed to the PARG null cell phenotype in response to genotoxic stress.

The Saccharomyces cerevisiae ySwi/Snf-catalyzed formation of nucleosome dimers is described by using mono- and dinucleosome templates assembled from purified histones and DNA of the high-affinity (601) nucleosome positioning sequence. The effects of nucleosome template geometry on the formation of altered dimers and assayed formation of altered nucleosome pairs on reconstituted dinucleosomes are evaluated.

The first X-ray crystal structures of [Co.sup.2+], [Fe.sup.2+] and [Mn.sup.2+] metal-substituted human histone deacetylases (HDACs), each complexed with the inhibitor M344 is reported. The results provide insight into the metal dependence of the substrate [K.sub.M] values and the [K.sub.i] values of hydroxamate inhibitors that chelate the active site metal and the affinities of metal ions that are comparable to the readily exchangeable metal concentration estimated in living cells.

The metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) is examined and a second [Mg.sup.2+] in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. The reaction has followed the same rapid equilibrium random mechanism in the presence or absence of the second [Mg.sup.2+], which is observed by initial velocity analysis and substrate analogue and product inhibition studies.

The kinetic modeling of H1 recovery curves obtained during fluorescence recovery after photobleaching (FRAP) experiments are utilized to determine the impact of core histone acetylation on the different variants of H1. The hyperacetylation of core histones are found to affect the cooperative nature of low-affinity H1 binding, with some variants undergoing a predicted decrease of almost 2 orders of magnitude.

Tritium as a radioactive label was used to develop a new footprinting method for mapping protein interactions. The application of developed footprinting method to the complex formed by the histone H3 fragment [H3.sub.122-135] and the protein [hAsf1A.sub.1-156] afforded data in good agreement with NMR results.

The synthetically curved DNA segments, 108, 180, and 252 bp were used to understand the underlying superhelically curved DNA mediated activation mechanism in yeast cells. The finding from the study provides important guidelines for designing and constructing artificial chromatin modulators as a tool for chromatin engineering.

The small molecule versus peptide relationships in LSD1 inhibition is understood by furthering the analysis of warheads in peptide scaffolds to include the chlorovinyl, endo-cyclopropylamine and hydrazine-functionalities as LSD1 inactivators. The studies have shown that phenelzine has blocked H3K4Me demethylation in cells, which is validated as a pharmacologic tool and potential lead structure for anticancer therapy.

Several biochemical and biophysical studies are conducted to explain the properties, as well as the structural role played by the H2A.Z and H3.3 histone variants. These histones are shown to be widely involved in the creation of new positions and the alteration of relative occupancy of the existing nucleosome position space.