Histone Deacetylase HDA6 Research Articles

Histone deacetylase HDA6 suppresses intergenic transcription and mediates crosstalk between DNA methylation and histone modifications in rRNA gene silencing

Histone deacetylase HDA6 suppresses intergenic transcription and mediates crosstalk between DNA methylation and histone modifications in rRNA gene silencing

Involvement of Arabidopsis histone deacetylase HDA6 in ABA and salt stress response

Histone modifications play an important role in the epigenetic regulation of gene expression. All histone modifications are reversible, which may therefore provide a flexible way for regulating gene expression during the plant's development and during the plant response to environmental stimuli. The reversible acetylation and deacetylation of specific lysine residues on core histones are catalysed by histone acetyltransferases and histone deacetylases (HDAs). HDA6 is an RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering plants displayed a phenotype that was hypersensitive to ABA and salt stress. Compared with wild-type plants, the expression of the ABA and abiotic stress-responsive genes, ABI1, ABI2, KAT1, KAT2, DREB2A, RD29A, and RD29B, was decreased in axe1-5 and HDA6 RNA-interfering plants when treated with ABA or salt stress. It was found that both ABA and salt stress could enrich the gene activation markers, histone H3K9K14 acetylation, and H3K4 trimethylation, but decrease the gene repression marker, H3K9 dimethylation, of the ABA and abiotic stress-responsive genes. Our study indicates that HDA6-involved histone modifications modulate seed germination and the salt stress response, as well as ABA- and salt stress-induced gene expression in Arabidopsis.

Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.

Phospholipase C is required for histone acetylation in Saccharomyces cerevisiae

Phospholipase C (Plc1p) is required for the initial step of inositol polyphosphates (InsPs) synthesis and yeast cells with deletion of PLC1 gene are completely devoid of any InsPs and display aberrations in transcriptional regulation. To address the mechanism how InsPs affect transcriptional regulation, we have utilized inducible FLR1 promoter, which depends on Plc1p for activation. We show that Plc1p is required for normal level of histone H3 acetylation, recruitment of Swi/Snf and SAGA complexes, and nucleosome eviction in the FLR1 promoter. In addition, plc1Δ cells display decreased cellular levels of acetylated histones H3, H4, and histone H2A variant Htz1p. In accordance with the role of Plc1p in supporting histone acetylation, plc1Δ mutation is synthetically lethal with mutations in several subunits of SAGA and NuA4 histone acetyltransferase complexes. Moreover, the growth rate, sensitivity to multiple stresses, and the transcriptional defects of plc1Δ cells are partially suppressed by deletion of histone deacetylase HDA1. The results thus provide evidence that Plc1p and InsPs are important for normal level of histone acetylation and that InsPs deprivation results in histone hypoacetylation that negatively affects number of physiological processes in plc1Δ cells.

Histone modifying proteins Gcn5 and Hda1 affect flocculation in Saccharomyces cerevisiae during high-gravity fermentation

The performance of yeast is often limited by the constantly changing environmental conditions present during high-gravity fermentation. Poor yeast performance contributes to incomplete and slow utilization of the main fermentable sugars which can lead to flavour problems in beer production. The expression of the FLO and MAL genes, which are important for the performance of yeast during industrial fermentations, is affected by complex proteins associated with Set1 (COMPASS) resulting in the induction of flocculation and improved maltose fermentation capacity during the early stages of high-gravity fermentation. In this study, we investigated a possible role for other histone modifying proteins. To this end, we tested a number of histone deacetylases (HDACs) and histone acetyltransferases and we report that flocculation is induced in absence of the histone deacetylase Hda1 or the histone acetyltransferase Gcn5 during high-gravity fermentation. The absence of Gcn5 protein also improved utilization of high concentrations of maltose. Deletion of SIR2 encoding the HDA of the silent informator regulator complex, did not affect flocculation under high-gravity fermentation conditions. Despite the obvious roles for Hda1 and Gcn5 in flocculation, this work indicates that COMPASS mediated silencing is the most important amongst the histone modifying components to control the expression of the FLO genes during high-gravity fermentation.

Structural and Functional Studies of the Yeast Class II Hda1 Histone Deacetylase Complex

Yeast class II Hda1 histone deacetylase (HDAC) complex is an H2B- and H3-specific HDAC in Saccharomyces cerevisiae consisting of three previously identified subunits, the catalytic subunit scHda1p and two non-catalytic structural subunits scHda2p and scHda3p. We co-expressed and co-purified recombinant yeast class II HDAC complex from bacteria as a functionally active and trichostatin-A-sensitive hetero-tetrameric complex. According to an extensive analysis of domain organization and interaction of all subunits (or domains), the N-terminal domain of scHda1p associates through the C-terminal coiled-coil domains (CCDs) of the scHda2p–scHda3p sub-complex, yielding a truncated scHda1pHDAC–scHda2pCCD2–scHda3pCCD3 complex with indistinguishable deacetylase activity compared to the full-length complex in vitro. We characterized the interaction of the HDAC complex with either single-stranded or double-stranded DNA and identified the N-terminal halves of scHda2p and scHda3p as binding modules. A high-resolution structure of the scHda3p DNA-binding domain by X-ray crystallography is presented. The crystal structure shows an unanticipated structural homology with the C-terminal helicase lobes of SWI2/SNF2 chromatin-remodeling domains of the Rad54 family enzymes. DNA binding is unspecific for nucleotide sequence and structure, similar to the Rad54 enzymes in vitro. Our structural and functional analyses of the budding yeast class II Hda1 HDAC complex provide insight into DNA recognition and deacetylation of histones in nucleosomes.

Basal Defense in Arabidopsis: WRKYs Interact with Histone Deacetylase HDA19

Plants face a diverse array of pathogen attack strategies in the wild, and regulation of the defense response often is a matter of life or death. WRKY transcription factors regulate the expression of many defense genes (reviewed in [Eulgem and Somssich, 2007][1]); in Arabidopsis , activation of

Antisense RNA Stabilization Induces Transcriptional Gene Silencing via Histone Deacetylation in S. cerevisiae

Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications.

The PLZF-like Protein TRA-4 Cooperates with the Gli-like Transcription Factor TRA-1 to Promote Female Development in C. elegans

The Gli-like transcription factor TRA-1 of C. elegans promotes female development by repressing the transcription of male-specific genes. We have found that tra-1 interacts with tra-4, a previously uncharacterized gene that encodes a protein similar to the human proto-oncoprotein and transcriptional repressor PLZF. In this context, the TRA-4 protein functions with NASP-1, a C. elegans homolog of the mammalian histone chaperone NASP, and the histone deacetylase HDA-1. We also found that tra-4 is a member of the synMuv B group of genes, many of which encode homologs of components of the Drosophila Myb-Muv B transcriptional repressor complex, and that several synMuv B genes also promote female development. Based on these results, we propose that male-specific genes are repressed in C. elegans hermaphrodites by the combined action of TRA-1/Gli, a complex composed of TRA-4/PLZF-like, NASP, and HDA-1/HDAC, and synMuv B proteins. Similar interactions may function in sex determination and developmental regulation in other species.

Suppressor Analysis of a Histone Defect Identifies a New Function for the Hda1 Complex in Chromosome Segregation

Histones are essential for the compaction of DNA into chromatin and therefore participate in all chromosomal functions. Specific mutations in HTA1, one of the two Saccharomyces cerevisiae genes encoding histone H2A, have been previously shown to cause chromosome segregation defects, including an increase in ploidy associated with altered pericentromeric chromatin structure, suggesting a role for histone H2A in kinetochore function. To identify proteins that may interact with histone H2A in the control of ploidy and chromosome segregation, we performed a genetic screen for suppressors of the increase-in-ploidy phenotype associated with one of the H2A mutations. We identified five genes, HHT1, MKS1, HDA1, HDA2, and HDA3, four of which encode proteins directly connected to chromatin function: histone H3 and each of the three subunits of the Hda1 histone deacetylase complex. Our results show that Hda3 has functions distinct from Hda2 and Hda1 and that it is required for normal chromosome segregation and cell cycle progression. In addition, HDA3 shows genetic interactions with kinetochore components, emphasizing a role in centromere function, and all three Hda proteins show association with centromeric DNA. These findings suggest that the Hda1 deacetylase complex affects histone function at the centromere and that Hda3 has a distinctive participation in chromosome segregation. Moreover, these suppressors provide the basis for future studies regarding histone function in chromosome segregation.

The C. elegans histone deacetylase HDA-1 is required for cell migration and axon pathfinding

Histone proteins play integral roles in chromatin structure and function. Histones are subject to several types of posttranslational modifications, including acetylation, which can produce transcriptional activation. The converse, histone deacetylation, is mediated by histone deacetylases (HDACs) and often is associated with transcriptional silencing. We identified a new mutation, cw2, in the Caenorhabditis elegans hda-1 gene, which encodes a histone deacetylase. Previous studies showed that a mutation in hda-1, e1795, or reduction of hda-1 RNA by RNAi causes defective vulval and gonadal development leading to sterility. The hda-1(cw2) mutation causes defective vulval development and reduced fertility, like hda-1(e1795), albeit with reduced severity. Unlike the previously reported hda-1 mutation, hda-1(cw2) mutants are viable as homozygotes, although many die as embryos or larvae, and are severely uncoordinated. Strikingly, in hda-1(cw2) mutants, axon pathfinding is defective; specific axons often appear to wander randomly or migrate in the wrong direction. In addition, the long range migrations of three neuron types and fasciculation of the ventral nerve cord are defective. Together, our studies define a new role for HDA-1 in nervous system development, and provide the first evidence for HDAC function in regulating neuronal axon guidance.

Involvement of Putative SNF2 Chromatin Remodeling Protein DRD1 in RNA-Directed DNA Methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals [1, 2]. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter [3], as well as the DMTase MET1 [4] and the putative histone deacetylase HDA6 [5] in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 [6, 7] and ATRX [8, 9], which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.

Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats.

Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

Regulation and processing of maize histone deacetylase Hda1 by limited proteolysis.

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases.

Cloning and structural characterization of the human histone deacetylase 6 gene

Histone deacetylases (HDACs) play a central role in the modification of chromatin structure and thus in the regulation of transcription and cellular differentiation. Based on structural and functional similarities, mammalian histone deacetylases may be grouped into three categories, class I HDACs, which are homologs of the yeast histone deacetylase RPD3, class II HDACs, which share a significant degree of homology with the yeast histone deacetylase HDA1 and class III HDACs which are closely related to the yeast SIR2 protein. We have isolated and characterized the human HDAC6 genomic sequence, which spans a region of 21,923 bp and which has one single genomic locus. Determination of the exon-intron splice junctions established that HDAC6 is encoded by 28 exons ranging in size from 41 bp (exon 5) to 677 bp (exon 24). Characterization of the 5' flanking genomic region, which precedes the HDAC6 open reading frame, revealed a TATA- and CCAAT-boxless promoter that contains a 1 kb CpG island. The 3,648 bp human HDAC6 mRNA encodes a 1,215 aa protein with a predictive molecular weight of 131.4 kDa. Fluorescence in situ hybridization analysis localized the human HDAC6 gene to the subband border of chromosome Xp11.22-23, a region which is characterized by frequent gains and losses of chromosomal material in several types of cancer and neurological disorders.

Histone deacetylase Hda1 acts as repressor of the Ustilago maydis biotrophic marker gene mig1

The Ustilago maydis mig1 gene is extensively up-regulated during growth within its host plant. A genetic approach was set up to identify mutants expressing mig1 during axenic growth. Five independent mutants were identified that not only displayed increased transcript levels of mig1 but also of egl1, an endoglucanase expressed in dikaryotic filaments. egl1 has recently been shown to be repressed by Hda1, a putative histone deacetylase [Reichmann et al., submitted]. The identified UV mutants shared other phenotypes with hda1 deletion mutants like enhanced pigmentation and the inability to produce teliospores in maize tumours. Complementation and sequence analysis demonstrated that all five UV mutants contained point mutations in the hda1 gene. Despite a common repression mechanism, expression levels of mig1 and egl1 were significantly different during axenic and biotrophic growth, providing evidence for additional regulatory inputs from the respective growth stage. Furthermore, while egl1 is subject to repression by the U. maydis regulator Rum1, this was not the case for mig1. U. maydis strains deleted in either hda1 or rum1 were not affected in mig1 expression in the tumour stage. Transcript levels conferred by mig1 promoters deleted in negatively cis-acting sequences exceeded those in hda1 mutants, suggesting additional negative factors governing mig1 expression.

The histone deacetylase Hda1 from Ustilago maydis is essential for teliospore development

In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. It is thought that several of the b-regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b-dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild-type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Deltahda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.

A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during Caenorhabditis elegans embryogenesis.

In Caenorhabditis elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. In this study, we show that CBP-1, HDA-1 and POP-1 converge on end-1, an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appears to be the activation of end-1 transcription. We further identify a molecular mechanism for the endoderm-suppressive effect of POP-1 in the MS lineage by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. We provide evidence that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C.elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription.

HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1

Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo. In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits.

TUP1 Utilizes Histone H3/H2B–Specific HDA1 Deacetylase to Repress Gene Activity in Yeast

TUP1 is recruited to and represses genes that regulate mating, glucose and oxygen use, stress response, and DNA damage. It is shown here that disruption of either TUP1 or histone deacetylase HDA1 causes histone H3/H2B–specific hyperacetylation next to the TUP1 binding site at the stress-responsive ENA1 promoter. It is also shown that TUP1 interacts with HDA1 in vitro. These data indicate that TUP1 mediates localized histone deacetylation through HDA1. Interestingly, RPD3 deacetylates the ENA1 coding region, and both deacetylases contribute to ENA1 repression. However, epistasis analysis argues that only HDA1 and TUP1 are likely to function in the same pathway. These data define gene and histone targets of HDA1 and illustrate the role of histone deacetylation in TUP1 repression.