Complete field-induced spectral response of the spin-1/2 triangular-lattice antiferromagnet CsYbSe2.

Fifty years after Anderson's resonating valence-bond proposal, the spin-1/2 triangular-lattice Heisenberg antiferromagnet (TLHAF) remains the ultimate platform to explore highly entangled quantum spin states in proximity to magnetic order. Yb-based delafossites are ideal candidate TLHAF materials, which allow experimental access to the full range of applied in-plane magnetic fields. We perform a systematic neutron scattering study of CsYbSe2, first proving the Heisenberg character of the interactions and quantifying the second-neighbor coupling. We then measure the complex evolution of the excitation spectrum, finding extensive continuum features near the 120°-ordered state, throughout the 1/3-magnetization plateau and beyond this up to saturation. We perform cylinder matrix-product-state (MPS) calculations to obtain an unbiased numerical benchmark for the TLHAF and spectacular agreement with the experimental spectra. The measured and calculated longitudinal spectral functions reflect the role of multi-magnon bound and scattering states. These results provide valuable insight into unconventional field-induced spin excitations in frustrated quantum materials.

Acf1, the largest subunit of CHRAC, regulates ISWI-induced nucleosome remodelling.

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.

Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3[middle dot]Rpd3 histone deacetylase complex and is required for histone deacetylase activity.

SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background. In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex.

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.

Two histone fold proteins, CHRAC-14 and CHRAC-16, are developmentally regulated subunits of chromatin accessibility complex (CHRAC).

The ISWI ATPase of Drosophila is a molecular engine that can drive a range of nucleosome remodelling reactions in vitro. ISWI is important for cell viability, developmental gene expression and chromosome structure. It interacts with other proteins to form several distinct nucleosome remodelling machines. The chromatin accessibility complex (CHRAC) is a biochemical entity containing ISWI in association with several other proteins. Here we report on the identification of the two smallest CHRAC subunits, CHRAC-14 and CHRAC-16. They contain histone fold domains most closely related to those found in sequence-specific transcription factors NF-YB and NF-YC, respectively. CHRAC-14 and CHRAC-16 interact directly with each other as well as with ISWI, and are associated with functionally active CHRAC. The developmental expression profiles of both subunits suggest specialized roles in chromatin remodelling reactions in the early embryo for both histone fold subunits.

Analysis of the histone acetyltransferase B complex of maize embryos.

Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45. Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases. Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap. Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45. Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination. Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA. Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.

The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae.

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.

Expanded lysine acetylation specificity of Gcn5 in native complexes.

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes.

Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays.

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

Identification and analysis of yeast nucleosomal histone acetyltransferase complexes.

Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin. New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT). The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation. However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones. It is therefore very likely that additional proteins are required for Gcn5-mediated acetylation of chromosomal histones. We have recently shown that Gcn5 is the catalytic subunit of two high-molecular-weight histone acetyltransferase complexes in yeast. In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast. These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively. Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae. These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

Transcriptional activators direct histone acetyltransferase complexes to nucleosomes.

Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5, as well as human p300/CBP, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4-VP16-bound nucleosomes for acetylation. We find that Gal4-VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

A comparative study of histone deacetylases of plant, fungal and vertebrate cells.

The enzymatic equilibrium of reversible core histone acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase (HD). These enzyme activities exist as multiple enzyme forms. The present report describes methods to extract different HD-forms from three organisms, germinating maize embryos, the myxomycete Physarum polycephalum, and chicken red blood cells; it provides data on the chromatographic separation and partial purification of HD-forms. In germinating maize embryos three HDs (HD1-A, HD1-B, HD2) can be discriminated; HD1-A, HD1-B, and HD2 were characterized in terms of their dependence on pH, temperature and various ions, as well as kinetic parameters (Km for core histones) and inhibition by various compounds. The same parameters were investigated for the corresponding enzymes of Physarum polycephalum, and mature and immature chicken erythrocytes. Based on these results, optimum assay conditions were established for the different enzyme forms. The kinetic data revealed that the maize histone deacetylase HD1-B peak after partial purification by Q-Sepharose chromatography was heterogeneous and consisted of two histone binding sites that differed significantly in their affinity for purified core histones. Optimized affinity chromatography on poly-Lysine Agarose indeed showed that the former defined deacetylase HD1-B can be separated clearly into two individual HD enzyme forms. The high multiplicity of histone deacetylases underlines the importance of these enzymes for the complex regulation of core histone acetylation.

Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos.

From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.

Nuclear matrix and the cell cycle.

The facts that the nuclear matrix represents a structural framework of the cell nucleus and that nuclear events, such as DNA replication, transcription, and DNA repair, are associated with this skeletal structure suggest that its components are subject to cell cycle-regulatory mechanisms. Cell cycle regulation has been shown for nuclear lamina assembly and disassembly during mitosis and chromatin reorganization. Little attention has so far been paid to internal nuclear matrix proteins and matrix-associated proteins with respect to the cell cycle. This survey attempts to summarize available data and presents experimental evidence that important metabolic functions of the nucleus are regulated by the transient, cell cycle-dependent attachment of enzymes and regulatory proteins to the nuclear matrix. Results on thymidine kinase and RNA polymerase during the synchronous cell cycle of Physarum polycephalum demonstrate that reversible binding to the nuclear matrix represents an additional level of regulation for nuclear processes.

Subcellular location of enzymes involved in core histone acetylation.

Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos. We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination. The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination. In contrast, HD-2 was chromatin bound during the entire germination pathway. Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination. Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction. To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities. This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol. This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes. Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.

Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC-PK1 cell line. A comprehensive investigation of different preparation procedures.

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.