Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression

Little is known about the role of genetic and epigenetic control in the spatial and temporal regulation of plant development. Overexpressing antisense Arabidopsis thaliana HD1 (AtHD1) encoding a putative major histone deacetylase induces pleiotropic effects on plant growth and development. It is unclear whether the developmental abnormalities are caused by a defective AtHD1 or related homologs and are heritable in selfing progeny. We isolated a stable antisense AtHD1 (CASH) transgenic line and a T-DNA insertion line in exon 2 of AtHD1, resulting in a null allele (athd1-t1). Both athd1-t1 and CASH lines display increased levels of histone acetylation and similar developmental abnormalities, which are heritable in the presence of antisense AtHD1 or in the progeny of homozygous (athd1-t1/athd1-t1) plants. Furthermore, when the athd1-t1/athd1-t1 plants are crossed to wild-type plants, the pleiotropic developmental abnormalities are immediately restored in the F(1) hybrids, which correlates with AtHD1 expression and reduction of histone H4 Lys12 acetylation. Unlike the situation with the stable code of DNA and histone methylation, developmental changes induced by histone deacetylase defects are immediately reversible, probably through the restoration of a reversible histone acetylation code needed for the normal control of gene regulation and development.     

HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis

Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:beta-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and beta-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.     

Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis

Histone acetylation and deacetylation play an important role in epigenetic controls of gene expression. HISTONE DEACETYLASE6 (HDA6) is a REDUCED POTASSIUM DEPENDENCY3-type histone deacetylase, and the Arabidopsis (Arabidopsis thaliana) hda6 mutant axe1-5 displayed a late-flowering phenotype. axe1-5/flc-3 double mutants flowered earlier than axe1-5 plants, indicating that the late-flowering phenotype of axe1-5 was FLOWERING LOCUS C (FLC) dependent. Bimolecular fluorescence complementation, in vitro pull-down, and coimmunoprecipitation assays revealed the protein-protein interaction between HDA6 and the histone demethylase FLD. It was found that the SWIRM domain in the amino-terminal region of FLD and the carboxyl-terminal region of HDA6 are responsible for the interaction between these two proteins. Increased levels of histone H3 acetylation and H3K4 trimethylation at FLC, MAF4, and MAF5 were found in both axe1-5 and fld-6 plants, suggesting functional interplay between histone deacetylase and demethylase in flowering control. These results support a scenario in which histone deacetylation and demethylation cross talk are mediated by physical association between HDA6 and FLD. Chromatin immunoprecipitation analysis indicated that HDA6 bound to the chromatin of several potential target genes, including FLC and MAF4. Genome-wide gene expression analysis revealed that, in addition to genes related to flowering, genes involved in gene silencing and stress response were also affected in hda6 mutants, revealing multiple functions of HDA6. Furthermore, a subset of transposons was up-regulated and displayed increased histone hyperacetylation, suggesting that HDA6 can also regulate transposons through deacetylating histone.     

Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis

Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis (Arabidopsis thaliana), the histone acetyltransferase AtHAC1 is homologous to animal p300/CREB (cAMP-responsive element-binding protein)-binding proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. The functions of p300/CREB-binding proteins in animals are well characterized, whereas little is known about the roles of AtHAC1 in developmental control in Arabidopsis. Lesions in AtHAC1 caused pleiotropic developmental defects, including delayed flowering, a shortened primary root, and partially reduced fertility. Analysis of the molecular basis of late flowering in hac1 mutants showed that the hac1 plants respond normally to day length, gibberellic acid treatment, and vernalization. Furthermore, the expression level of the flowering repressor FLOWERING LOCUS C (FLC) is increased in hac1 mutants, indicating that the late-flowering phenotype of hac1 mutants is mediated by FLC. Since histone acetylation is usually associated with the activation of gene expression, histone modifications of FLC chromatin are not affected by mutations in HAC1 and expression levels of all known autonomous pathway genes are unchanged in hac1 plants, we propose that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC.     

HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis

Post-translational modifications of histones, including acetylation, play a key role in modulating dynamic changes in chromatin structure and gene activity. Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases. HDA6 is a RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering (HDA6-RNAi) plants displayed higher levels of acetylated H3 compared with wild-type, suggesting that HDA6 affects histone acetylation levels globally. The expression of the jasmonate responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in axe1-5 and HDA6-RNAi plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression. Our results suggest that HDA6 is involved in jasmonate response, senescence, and flowering in Arabidopsis.     

Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin. Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7. A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both Igf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles. A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains.     

The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms

FLOWERING LOCUS C (FLC), a repressor of flowering, is a major determinant of flowering time in Arabidopsis. FLC expression is repressed by vernalization and in plants with low levels of DNA methylation, resulting in early flowering. This repression is not associated with changes of DNA methylation within the FLC locus in either vernalized plants or plants with low levels of DNA methylation. In both cases, there is a reduction of histone H3 trimethyl-lysine 4 (K4) and acetylation of both histones H3 and H4 around the promoter-translation start of FLC. The expression of the two genes flanking FLC is also repressed in both conditions and repression is associated with decreased histone H3 acetylation. The changes in histone modifications at the FLC gene cluster, which are similar in vernalized plants and in plants with reduced DNA methylation, must arise by different mechanisms. VERNALIZATION 1, VERNALIZATION 2 and VERNALIZATION INSENSITIVE 3 modulate FLC expression in vernalized plants; these proteins play no role in the downregulation of FLC in plants with low levels of DNA methylation. Chimeric FLC::GUS transgenes respond to vernalization but these same transgenes show a position-dependent response to low levels of DNA methylation. In plants with reduced DNA methylation, expression of the five MADS AFFECTING FLOWERING (MAF) genes is repressed, suggesting that DNA methylation alters the expression of a trans-acting regulator common to FLC and members of the related MAF gene family. Our observations suggest that DNA methylation is not part of the vernalization pathway.