Mechanism Of Histone Deacetylases And Their Associated Proteins In Regulating Flowering Time

In eukaryotes,gene expression is controlled not only by DNA sequences but also by chromatin modifications such as DNA methylation and histone modifications.Histone modifications play critical roles in the regulation of flowering time.Previous studies showed that the closely related Arabidopsis histone deacetylases HDA6 and HDA19 are involved in the regulation of flowering time.However,the molecular mechanisms underlying the regulation are poorly understood.This study identified proteins co-purified with HDA6 by affinity purification in combination with mass spectrometry,and indicated that HDC1,Sin3-like proteins(SNLs),and MSI1 can interact with HDA6 and form a histone deacetylase complex which is similar to the conserved yeast RPD3-type histone deacetylase complex.In addition,we found that HDA19 can also interact with HDC1,SNLs,and MSI1 by identifing proteins co-purified with HDC1 and SNL3 through the same method,forming an RPD3-type histone deacetylase complex.Previous studies indicated that HDA6 is required for DNA methylation and heterochromatin silencing.In this study,Southern blotting and DNA methylation-sensitive enzyme digestion followed by PCR(chop-PCR)results showed that HDC1 is dispensable for the function of HDA6 in DNA methylation and transcriptional silencing.The hda6 mutant exhibited a late flowering phenotype not only in long days(LD)but also in short days(SD),indicating that HDA6 regulates flowering time through the autonomous pathway.Different from the hda6 mutant,the hdcl,snl2/3/4,and hdal9 mutant plants flowered later than the wild type in LD but flowered earlier than the wild type in SD,suggesting that HDC1,SNLs,and HDA19 are involved the regulation of flowering time in a photoperiod-dependent manner.Previous studies showed that the expression of the flowering repressor gene FLC was increased in the hda6 mutant,resulting in a late flowering phenotype.In the hda6/flc double mutant,the flowering time was restored to the wild type level,suggesting that HDA6 regulates flowering time by repressing the expression of FLC,However,in LD,the expression of FLC was not increased in the hdcl mutant and the late flowering phenotype of the hdcl mutant was not significantly affected by the flc mutation,suggesting that HDC1 regulates flowering time via a mechanism that is distinct from that of HDA6 which is denpendent on FLC.Further research reveals that HDC1 and HDA19 are involved in histone deacetylation and transcriptional repression of the flowering repressor genes GASA5 and GA20X6 in LD.In SD,the FT expression increased in the hdcl,hda19,and snl2/3/4 mutants relative to the wild type and the ft-11 mutation restored the flowering time of the ft-11/hdcl and ft-11/hda 19 double mutant to the wild type level,indicating that the early flowering phenotype of HDA19 complex mutants depends on increased expression of FT.CONSTANS(CO),an FT activator in the photoperiod pathway,is almost completely degraded in SD,resulting in the late flowering phenotype.The hdcl and hda19 mutants showed an early flowering phenotype in the co-11 mutant background in LD,further confirming the function of the HDA19 complex in the regulation of flowering time through a photoperiod-dependent pathway.RNA-seq and H3Ac ChIP-seq results showed that HDC1,HDA6 and HDA19 repressed the expression of many genes by histone deacetylation,which is consistent with their role as a subunit of the histone deacetylase complex.Although HDC1 is a shared subunit of the HDA19 and HDA6 histone deacetylase complexes,the effects on genes expression caused by HDC1 are more similar to HDA19 than to HDA6,indicating that HDC1 primarily acts as a subunit of the HDA19 complex in the regulation of flowering time.Together,this study demonstrates that HDC1,SNLs and other proteins can form multi-subunit histone deacetylase complexes with HDA6 or HDA19,but the functions of subunits in the same complex are not identical.HDC1 is not involved in the HDA6-mediated DNA methylation,heterochromatin silencing,and flowering time regulation.HDC1 acts as a subunit of the HDA19 complex to regulate flowering time in a photoperiod-dependent manner and enable flowering plants to survive and reproduce via maintaining their proper vegetative or reproductive growth in different seasons.