SWI3B and HDA6 interact and are required for transposon silencing in Arabidopsis.

Although the interplay of covalent histone acetylation/deacetylation and ATP-dependent chromatin remodelling is crucial for the regulation of chromatin structure and gene expression in eukaryotes, the underlying molecular mechanism in plants remains largely unclear. Here we show a direct interaction between Arabidopsis SWI3B, an essential subunit of the SWI/SNF chromatin-remodelling complex, and the RPD3/HDA1-type histone deacetylase HDA6 both in vitro and in vivo. Furthermore, SWI3B and HDA6 co-repress the transcription of a subset of transposons. Both SWI3B and HDA6 maintain transposon silencing by decreasing histone H3 lysine 9 acetylation, but increasing histone H3 lysine 9 di-methylation, DNA methylation and nucleosome occupancy. Our findings reveal that SWI3B and HDA6 may act in the same co-repressor complex to maintain transposon silencing in Arabidopsis.

A domesticated Harbinger transposase forms a complex with HDA6 and promotes histone H3 deacetylation at genes but not TEs in Arabidopsis.

In eukaryotes, histone acetylation is a major modification on histone N-terminal tails that is tightly connected to transcriptional activation. HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements (TEs) in Arabidopsis thaliana. HDA6 has been shown to participate in several complexes in plants, including a conserved SIN3 complex. Here, we uncover a novel protein complex containing HDA6, several Harbinger transposon-derived proteins (HHP1, SANT1, SANT2, SANT3, and SANT4), and MBD domain-containing proteins (MBD1, MBD2, and MBD4). We show that mutations of all four SANT genes in the sant-null mutant cause increased expression of the flowering repressors FLC, MAF4, and MAF5, resulting in a late flowering phenotype. Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes, TE silencing is unaffected in sant-null mutants. Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation. Collectively, our findings suggest that Harbinger transposon-derived SANT domain-containing proteins are required for histone deacetylation and flowering time control in plants.

Histone Deacetylase TaHDT701 Functions in TaHDA6-TaHOS15 Complex to Regulate Wheat Defense Responses to Blumeria graminis f.sp. tritici.

Powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt) leads to severe economic losses in bread wheat (Triticum aestivum L.). To date, only a few epigenetic modulators have been revealed to regulate wheat powdery mildew resistance. In this study, the histone deacetylase 2 (HD2) type histone deacetylase TaHDT701 was identified as a negative regulator of wheat defense responses to Bgt. Using multiple approaches, we demonstrated that TaHDT701 associates with the RPD3 type histone deacetylase TaHDA6 and the WD40-repeat protein TaHOS15 to constitute a histone deacetylase complex, in which TaHDT701 could stabilize the TaHDA6-TaHOS15 association. Furthermore, knockdown of TaHDT701, TaHDA6, and TaHOS15 resulted in enhanced wheat powdery mildew resistance, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex negatively regulates wheat defense responses to Bgt. Moreover, chromatin immunoprecipitation assays revealed that TaHDT701 could function in concert with TaHOS15 to recruit TaHDA6 to the promoters of defense-related genes such as TaPR1, TaPR2, TaPR5, and TaWRKY45. In addition, silencing of TaHDT701, TaHDA6, and TaHOS15 resulted in the up-regulation of TaPR1, TaPR2, TaPR5, and TaWRKY45 accompanied with increased histone acetylation and methylation, as well as reduced nucleosome occupancy, at their promoters, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex suppresses wheat powdery mildew resistance by modulating chromatin state at defense-related genes.

Histone deacetylases HDA6 and HDA9 coordinately regulate valve cell elongation through affecting auxin signaling in Arabidopsis.

Both Histone Deacetylases HDA6 and HDA9 belong to class I subfamily of RPD3/HDA1 HDACs. Loss-of-function mutants of HDA9 form slightly blunt siliques. However, the involvement of HDA6 in regulating silique tip growth is unclear. In this study, we show that HDA6 acts redundantly with HDA9 in regulating the elongation of valve cells in the silique tip. Although the hda6 single mutant does not exhibit a detectable silique phenotype, the silique tip of hda6 hda9 double mutant displays a more severe bulge, a morphology we termed as "nock-shaped". The valve cells of the silique tip of hda9 are longer than wild-type, and loss of HDA6 in hda9 enhances the valve cell elongation phenotype. The transcript levels of auxin-signaling-related genes are mis-regulated in hda9 and hda6 hda9 siliques, and the GFP reporter driven by the auxin response promoter DR5 is weaker in hda9 or hda6 hda9 than wild-type or hda6. Thus, our findings reveal that HDA6 and HDA9 coordinately control the elongation of silique valve cells through regulating the expression of auxin-related genes in silique tips.

Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants.

Cytosine DNA methylation is a conserved epigenetic silencing mechanism that defends against biotic stresses such as geminivirus infection. As a countermeasure, geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Previous studies showed that V2 protein of Tomato yellow leaf curl virus (TYLCV) functions as a TGS suppressor. However, how V2 mediates TGS suppression remains unknown. Here we show that V2 interacts directly with a Nicotiana benthamiana histone deacetylase 6 (NbHDA6), a homolog of Arabidopsis HDA6 (AtHDA6), known to be involved in gene silencing in cooperation with methyltransferase 1 (MET1). NbHDA6 genetically complemented a late-flowering phenotype and restored histone deacetylation of an AtHDA6 mutant. Furthermore, our investigation showed that NbHDA6 displayed histone deacetylase enzymatic activity, which was not inhibited by V2. Genetic analysis revealed that silencing of NbHDA6 expression resulted in enhanced susceptibility to TYLCV infection. In addition, methylation-sensitive PCR and bisulfite sequencing analysis showed that silencing of NbHDA6 expression caused reduced DNA methylation of the viral genome in infected plants. HDA6 was previously shown to recruit and physically interact with MET1 to function in gene silencing. Using competitive pulldown and coimmunoprecipitation assays, we demonstrated that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. These findings suggest that V2 interacts with host HDA6 and interferes with the recruitment of MET1 by HDA6, resulting in decreased methylation of the viral DNA genome by TGS with a concomitant increase in host susceptibility to TYLCV infection.IMPORTANCE Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses. In turn, geminiviruses encode proteins that inhibit methylation by TGS. Previous studies showed that TYLCV V2 can efficiently suppress TGS, but the mechanism remains unknown. We showed that V2 interacted with NbHDA6 but did not inhibit its enzymatic activity. As HDA6 is known to be involved in gene silencing in cooperation with MET1, we explored the relationship between V2, NbMET1, and NbHDA6. Our investigation showed that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. To our knowledge, this is the first report that viral proteins inhibit TGS by interacting with histone deacetylase but not by blocking the methyl cycle. This work provides an additional mechanism for TGS suppression by geminiviruses.

Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase.

Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development.

HISTONE DEACETYLASE 6 represses pathogen defence responses in Arabidopsis thaliana.

Plant defence mechanisms are suppressed in the absence of pathogen attack to prevent wasted energy and growth inhibition. However, how defence responses are repressed is not well understood. Histone deacetylase 6 (HDA6) is a negative regulator of gene expression, and its role in pathogen defence response in plants is not known. In this study, a novel allele of hda6 (designated as shi5) with spontaneous defence response was isolated from a forward genetics screening in Arabidopsis. The shi5 mutant exhibited increased resistance to hemibiotrophic bacterial pathogen Pst DC3000, constitutively activated expression of pathogen-responsive genes including PR1, PR2, etc. and increased histone acetylation levels at the promoters of most tested genes that were upregulated in shi5. In both wild type and shi5 plants, the expression and histone acetylation of these genes were upregulated by pathogen infection. HDA6 was found to bind to the promoters of these genes under both normal growth conditions and pathogen infection. Our research suggests that HDA6 is a general repressor of pathogen defence response and plays important roles in inhibiting and modulating the expression of pathogen-responsive genes in Arabidopsis.

Regulation of flowering time by the histone deacetylase HDA5 in Arabidopsis.

The acetylation level of histones on lysine residues regulated by histone acetyltransferases and histone deacetylases plays an important but under-studied role in the control of gene expression in plants. With the aim of characterizing the Arabidopsis RPD3/HDA1 family histone deacetylase HDA5, we present evidence showing that HDA5 displays deacetylase activity. Mutants defective in the expression of HDA5 displayed a late-flowering phenotype. Expression of the flowering repressor genes FLC and MAF1 was up-regulated in hda5 mutants. Furthermore, the gene activation markers, histone H3 acetylation and H3K4 trimethylation on FLC and MAF1 chromatin were increased in hda5-1 mutants. Chromatin immunoprecipitation analysis showed that HDA5 binds to the chromatin of FLC and MAF1. Bimolecular fluorescence complementation assays and co-immunoprecipitation assays showed that HDA5 interacts with FVE, FLD and HDA6, indicating that these proteins are present in a protein complex involved in the regulation of flowering time. Comparing gene expression profiles of hda5 and hda6 mutants by RNA-seq revealed that HDA5 and HDA6 co-regulate gene expression in multiple development processes and pathways.

ESR2-HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture.

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2-HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

Regulation of jasmonate signaling by reversible acetylation of TOPLESS in Arabidopsis.

The plant hormone jasmonate (JA) regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program. Key regulators of JA-responsive gene expression include the master transcription factor MYC2, which is repressed by the conserved Groucho/Tup1-like corepressor TOPLESS (TPL) in the resting state. However, the mechanisms underlying TPL-mediated transcriptional repression of MYC2 activity and hormone-dependent switching between repression and de-repression remain enigmatic. Here, we report the regulation of TPL activity and JA signaling by reversible acetylation of TPL. We found that the histone acetyltransferase GCN5 could mediate TPL acetylation, which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ (NINJA) adaptor and promotes its recruitment to MYC2 target promoters, facilitating transcriptional repression. Conversely, TPL deacetylation by the histone deacetylase HDA6 weakens TPL-NINJA interaction and inhibits TPL recruitment to MYC2 target promoters, facilitating transcriptional activation. In the resting state, the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis, promoting transcriptional repression activity of TPL. In response to JA elicitation, HDA6 expression is transiently induced, resulted in decreased TPL acetylation and repressor activity, thereby transcriptional activation of MYC2 target genes. Thus, the GCN5-TPL-HDA6 module maintains the homeostasis of acetylated TPL, thereby determining the transcriptional state of JA-responsive genes. Our findings uncovered a mechanism by which the TPL corepressor activity in JA signaling is actively tuned in a rapid and reversible manner.

Arabidopsis RPD3-like histone deacetylases form multiple complexes involved in stress response.

The Arabidopsis thaliana RPD3-type histone deacetylases have been known to form conserved SIN3-type histone deacetylase complexes, but whether they form other types of complexes is unknown. Here, we perform affinity purification followed by mass spectrometry and demonstrate that the Arabidopsis RPD3-type histone deacetylases HDA6 and HDA19 interact with several previously uncharacterized proteins, thereby forming three types of plant-specific histone deacetylase complexes, which we named SANT, ESANT, and ARID. RNA-seq indicates that the newly identified components function together with HDA6 and HDA19 and coregulate the expression of a number of genes. HDA6 and HDA19 were previously thought to repress gene transcription by histone deacetylation. We find that the histone deacetylase complexes can repress gene expression via both histone deacetylation-dependent and -independent mechanisms. In the mutants of histone deacetylase complexes, the expression of a number of stress-induced genes is up-regulated, and several mutants of the histone deacetylase complexes show severe retardation in growth. Considering that growth retardation is thought to be a trade-off for an increase in stress tolerance, we infer that the histone deacetylase complexes identified in this study prevent overexpression of stress-induced genes and thereby ensure normal growth of plants under nonstress conditions.

Arabidopsis SUMO E3 Ligase SIZ1 Interacts with HDA6 and Negatively Regulates HDA6 Function during Flowering.

The changes in histone acetylation mediated by histone deacetylases (HDAC) play a crucial role in plant development and response to environmental changes. Mammalian HDACs are regulated by post-translational modifications (PTM), such as phosphorylation, acetylation, ubiquitination and small ubiquitin-like modifier (SUMO) modification (SUMOylation), which affect enzymatic activity and transcriptional repression. Whether PTMs of plant HDACs alter their functions are largely unknown. In this study, we demonstrated that the Arabidopsis SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (SIZ1) interacts with HISTONE DEACETYLASE 6 (HDA6) both in vitro and in vivo. Biochemical analyses indicated that HDA6 is not modified by SUMO1. Overexpression of HDA6 in siz1-3 background results in a decreased level of histone H3 acetylation, indicating that the activity of HDA6 is increased in siz1-3 plants. Chromatin immunoprecipitation (ChIP) assays showed that SIZ1 represses HDA6 binding to its target genes FLOWERING LOCUS C (FLC) and MADS AFFECTING FLOWERING 4 (MAF4), resulting in the upregulation of FLC and MAF4 by increasing the level of histone H3 acetylation. Together, these findings indicate that the Arabidopsis SUMO E3 ligase SIZ1 interacts with HDA6 and negatively regulates HDA6 function.

Flavones and flavonols may have clinical potential as CK2 inhibitors in cancer therapy.

The serine-threonine kinase CK2, which targets over 300 cellular proteins, is overexpressed in all cancers, presumably reflecting its ability to promote proliferation, spread, and survival through a wide range of complementary mechanisms. Via an activating phosphorylation of Cdc373, a co-chaperone which partners with Hsp90, CK2 prolongs the half-life of protein kinases that promote proliferation and survival in many cancers, including Akt, Src, EGFR, Raf-1, and several cyclin-dependent kinases. CK2 works in other ways to boost the activity of signaling pathways that promote cancer aggressiveness and chemoresistance, including those driven by Akt, NF-kappaB, hypoxia-inducible factor-1, beta-catenin, TGF-beta, STAT3, hedgehog, Notch1, and the androgen receptor; it promotes the epidermal-mesenchymal transition and aids efficiency of DNA repair. Several potent and relatively specific inhibitors of CK2 are now being evaluated as potential cancer drugs; CX-4945 has shown impressive activity in cell culture studies and xenograft models, and is now entering clinical trials. Moreover, it has long been recognized that the natural flavone apigenin can inhibit CK2, with a Ki near 1 µM; more recent work indicates that a range of flavones and flavonols, characterized by a planar structure and hydroxylations at the 7 and 4' positions - including apigenin, luteolin kaempferol, fisetin, quercetin, and myricetin - can inhibit CK2 with Ki s in the sub-micromolar range. This finding is particularly intriguing in light of the numerous studies demonstrating that each of these agents can inhibit the growth of cancer cells lines in vitro and of human xenografts in nude mice. These studies attribute the cancer-retardant efficacy of flavones/flavonols to impacts on a bewildering array of cellular targets, including those whose activities are boosted by CK2; it is reasonable to suspect that, at least in physiologically achievable concentrations, these agents may be achieving these effects primarily via CK2 inhibition. Inefficient absorption and rapid conjugation limit the bioefficacy of orally administered flavonoids; however, the increased extracellular beta-glucuronidase of many tumors may give tumors privileged access to glucuronidated flavonoids, and nanopartical technology can improve the bioavailability of these agents. Enzymatically modified isoquercitrin has particular promise as a delivery vehicle for quercetin. Hence, it may be worthwhile to explore the clinical potential of flavones/flavonols as CK2 inhibitors for cancer therapy.

The LDL1/2-HDA6 Histone Modification Complex Interacts With TOC1 and Regulates the Core Circadian Clock Components in Arabidopsis.

In Arabidopsis, the circadian rhythm is associated with multiple important biological processes and maintained by multiple interconnected loops that generate robust rhythms. The circadian clock central loop is a negative feedback loop composed of the core circadian clock components. TOC1 (TIMING OF CAB EXPRESSION 1) is highly expressed in the evening and negatively regulates the expression of CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL). CCA1/LHY also binds to the promoter of TOC1 and represses the TOC1 expression. Our recent research revealed that the histone modification complex comprising of LYSINE-SPECIFIC DEMETHYLASE 1 (LSD1)-LIKE 1/2 (LDL1/2) and HISTONE DEACETYLASE 6 (HDA6) can be recruited by CCA1/LHY to repress TOC1 expression. In this study, we found that HDA6, LDL1, and LDL2 can interact with TOC1, and the LDL1/2-HDA6 complex is associate with TOC1 to repress the CCA1/LHY expression. Furthermore, LDL1/2-HDA6 and TOC1 co-target a subset of genes involved in the circadian rhythm. Collectively, our results indicate that the LDL1/2-HDA6 histone modification complex is important for the regulation of the core circadian clock components.

Genome-Wide Analysis of Gene Regulatory Networks of the FVE-HDA6-FLD Complex in Arabidopsis.

FVE/MSI4 is a homolog of the mammalian RbAp48 protein. We found that FVE regulates flowering time by repressing FLC through decreasing histone H3K4 trimethylation and H3 acetylation. Furthermore, FVE interacts with the histone deacetylase HDA6 and the histone demethylase FLD, suggesting that these proteins may form a protein complex to regulate flowering time. To further investigate the function of the FVE-FLD-HDA6 complex, we compared the gene expression profiles of fve, fld, and hda6 mutant plants by using RNA-seq analysis. Among the mis-regulated genes found in fve plants, 51.8 and 36.5% of them were also mis-regulated in fld and hda6 plants, respectively, suggesting that FVE, HDA6, and FLD may regulate the gene expression in the same developmental processes in Arabidopsis. Gene ontology analysis revealed that among 383 genes co-regulated by FVE, HDA6, and FLD, 15.6% of them are involved in transcription, 8.2% in RNA metabolic process, 7.7% in response to abiotic stress, and 6.3% in hormone stimulus. Taken together, these results indicate that HDA6, FVE, and FLD co-regulate the gene expression in multiple development processes and pathways.

HOS1-mediated activation of FLC via chromatin remodeling under cold stress.

The Arabidopsis E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) has been shown to act as a negative regulator of cold responses by degrading the INDUCER OF CBF EXPRESSION 1 (ICE1) transcription factor through the ubiquitin/proteasome pathway. Notably, loss-of-function hos1 mutants exhibit early flowering, and the transcript level of the floral repressor FLOWERING LOCUS C (FLC) is downregulated in the mutants. However, it is largely unknown how HOS1 regulates FLC transcription. We found that HOS1 activates FLC transcription by inhibiting the activity of histone deacetylase 6 (HDA6) under cold stress. Cold temperatures induce the binding of HOS1 to FLC chromatin in an FVE-dependent manner. Cold-activated HOS1 promotes the dissociation of HDA6 from FLC chromatin, and the cold effects disappear in both hos1 and fve mutants. It is therefore clear that HOS1 regulates FLC transcription via chromatin remodeling, providing new insights into the signaling crosstalks between cold response and flowering time control.