Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.     

The histone methyltransferase SDG8 regulates shoot branching in Arabidopsis

Histone lysine methylation is an evolutionally conserved modification involved in determining chromatin states associated with gene activation or repression. Here we report that the Arabidopsis SET domain group 8 (SDG8) protein is a histone H3 methyltransferase involved in regulating shoot branching. Knockout mutations of the SDG8 gene markedly reduce the global levels of histone H3 trimethylation at lysines 9 and 36 as well as dimethylation at lysine 36. The sdg8 mutants produce more shoot branches than wild-type plants. The expression of SPS/BUS (supershoot/bushy), a repressor of shoot branching, is decreased in sdg8 mutants, while UGT74E2 (UDP-glycosyltransferase 74E2), a gene associated with increased shoot branching, is up-regulated in sdg8 mutants. The altered expression of SPS/BUS and UGT74E2 correlates with changed histone H3 methylation at these loci. These results suggest that SDG8 regulates shoot branching via controlling the methylation states of its target genes.     

Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis

SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants mono- and dimethyl H3K9, mono- and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.     

HD2C interacts with HDA6 and is involved in ABA and salt stress response in Arabidopsis

HD2 proteins are plant specific histone deacetylases. Four HD2 proteins, HD2A, HD2B, HD2C, and HD2D, have been identified in Arabidopsis. It was found that the expression of HD2A, HD2B, HD2C, and HD2D was repressed by ABA and NaCl. To investigate the function of HD2 proteins further, two HD2C T-DNA insertion lines of Arabidopsis, hd2c-1 and hd2c-3 were identified. Compared with wild-type plants, hd2c-1 and hd2c-3 plants displayed increased sensitivity to ABA and NaCl during germination and decreased tolerance to salt stress. These observations support a role of HD2C in the ABA and salt-stress response in Arabidopsis. Moreover, it was demonstrated that HD2C interacted physically with a RPD3-type histone deacetylase, HDA6, and bound to histone H3. The expression of ABA-responsive genes, ABI1 and ABI2, was increased in hda6, hd2c, and hda6/hd2c-1 double mutant plants, which was associated with increased histone H3K9K14 acetylation and decreased histone H3K9 dimethylation. Taken together, our results suggested that HD2C functionally associates with HDA6 and regulates gene expression through histone modifications.     

C2H2 zinc finger-SET histone methyltransferase is a plant-specific chromatin modifier

Histone modification represents a universal mechanism for regulation of eukaryotic gene expression underlying diverse biological processes from neuronal gene expression in mammals to control of flowering in plants. In animal cells, these chromatin modifications are effected by well-defined multiprotein complexes containing specific histone-modifying activities. In plants, information about the composition of such co-repressor complexes is just beginning to emerge. Here, we report that two Arabidopsis thaliana factors, a SWIRM domain polyamine oxidase protein, AtSWP1, and a plant-specific C2H2 zinc finger-SET domain protein, AtCZS, interact with each other in plant cells and repress expression of a negative regulator of flowering, FLOWERING LOCUS C (FLC) via an autonomous, vernalization-independent pathway. Loss-of-function of either AtSWP1 or AtCZS results in reduced dimethylation of lysine 9 and lysine 27 of histone H3 and hyperacetylation of histone H4 within the FLC locus, in elevated FLC mRNA levels, and in moderately delayed flowering. Thus, AtSWP1 and AtCZS represent two main components of a co-repressor complex that fine tunes flowering and is unique to plants.