Identification of histone methylation modifiers and their expression patterns during somatic embryogenesis in Hevea brasiliensis.
ABSTRACT: Histone methylation plays a crucial role in various biological processes, from heterochromatin formation to transcriptional regulation. Currently, no information is available regarding histone methylation modifiers in the important rubber-producing plant Hevea brasiliensis. Here, we identified 47 histone methyltransferase (HMT) genes and 25 histone demethylase (HDM) genes as possible members of the histone methylation modifiers in the rubber tree genome. According to the structural features of HMT and HDM, the HbHMTs were classified into two groups (HbPRMs and HbSDGs), the HbHDMs have two groups (HbLSDs and HbJMJs). Expression patterns were analyzed in five different tissues and at different phases of somatic embryogenesis. HbSDG10, 21, 25, 33, HbJMJ2, 18, 20 were with high expression at different phases of somatic embryogenesis. HbSDG10,14, 20, 21, 33 and HbPRMT4 were expressed highly in anther, HbSDG14, 20, 21, 22, 23, 33, 35 and HbPRMT1 HbJMJ7 and HbLSD1, 2, 3, 4 showed high expression levels in callus. HbSDG1, 7, 10, 13, 14, 18, 19, 21, 22, 23, 35, HbPRMT1, 8, HbJMJ5, 7, 11, 16, 20 and HbLSD2, 3, 4 were expressed highly in somatic embryo. HbSDG10, 21, 25, 33, HbLSD2, 3 were expressed highly in bud of regenerated plant. The analyses reveal that HbHMTs and HbHDMs exhibit different expression patterns at different phases during somatic embryogenesis, implying that some HbHMTs and HbHDMs play important roles during somatic embryogenesis. This study provide fundamental information for further studies on histone methylation in Hevea brasiliensis.
Project description:BACKGROUND: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. RESULTS: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. CONCLUSIONS: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.
Project description:In Arabidopsis, the rapid-flowering summer-annual versus the vernalization-requiring winter-annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter-annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)-specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36- and H3K4-specific histone demethylase (HDM) activities of autonomous-pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter-annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36-specific HMT and HDM activities.
Project description:Low temperature influences the development and latex production of rubber trees (Hevea brasiliensis) when extension to suboptimal high-latitude areas. The successful extension of Hevea brasiliensis cultivation to high-latitude areas has long believed to benefit from the breeding of cold-tolerant cultivars. A puzzling incongruity is the variation in cold tolerance among the cultivated clones despite their similar genetic make-up.To investigate this, we first transferred cultivar Reyan 7-33-97 to short-term cold treatment, and showed that cold-related genes (such as HbICE1 and HbCBF2), cold-responsive (COR) genes, and DNA-methylation related genes (such as HbMET1) were induced by cold treatment. Furthermore, long-term cold treatment not only elevated the transcriptional activities of the HbICE1, HbCBF2, and HbMET, but also induced DNA demethylation of their promoters. Cold treatment increased the transcriptional activities of demethylation-related genes such as the HbDME, HbROS, and HbDML genes, but did not alter the promoter methylation status. Furthermore, the HbICE1 and HbMET promoters showed hypomethylation status in samples collected at the end of winter from 12 different cultivars grown in four geographical locations, but switched to hypermethylation status at the end of summer. Expression of COR was correlated with the low temperature. Given that little genetic diversity exists in the HbICE1 and HbMET promoters among different cultivars, the DNA demethylation induced by cold was highly correlated with low temperature, but not with the genetic backgrounds of cultivars.Cold-induced epigenetic modification might play an important role in cold tolerance of H. brasiliensis.
Project description:Modifications to histones, including acetylation and methylation processes, play crucial roles in the regulation of gene expression in plant development as well as in stress responses. However, limited information on the enzymes catalyzing histone acetylation and methylation in non-model plants is currently available. In this study, several histone modifier (HM) types, including six histone acetyltransferases (HATs), 11 histone deacetylases (HDACs), 48 histone methyltransferases (HMTs), and 22 histone demethylases (HDMs), are identified in litchi (Litchi chinensis Sonn. cv. Feizixiao) based on similarities in their sequences to homologs in Arabidopsis (A. thaliana), tomato (Solanum lycopersicum), and rice (Oryza sativa). Phylogenetic analyses reveal that HM enzymes can be grouped into four HAT, two HDAC, two HMT, and two HDM subfamilies, respectively, while further expression profile analyses demonstrate that 17 HMs were significantly altered during fruit abscission in two field treatments. Analyses reveal that these genes exhibit four distinct patterns of expression in response to fruit abscission, while an in vitro assay was used to confirm the HDAC activity of LcHDA2, LcHDA6, and LcSRT2. Our findings are the first in-depth analysis of HMs in the litchi genome, and imply that some are likely to play important roles in fruit abscission in this commercially important plant.
Project description:CBP (CREB-binding protein) is involved in transcriptional activation by a great variety of sequence-specific transcription factors. CBP has been shown to activate transcription through its histone acetyl transferase activity. Acetylation is a common post-translational modification of nucleosomal histone N-terminal tails, which generally correlates with transcriptional activation. Histone N-terminal tails are also modified by methylation but its functional consequences are largely unknown. Here we found that immunoprecipitation of CBP, or of the highly related p300, led to the co-immunoprecipitation of a robust histone methyl transferase (HMT) activity, indicating that CBP physically interacts with an HMT in living cells. The CBP-associated HMT is specific for lysines 4 and 9 of histone H3, which are known to be methylated in living cells. These results suggest that histone methylation could be involved in transcriptional activation. Furthermore, they raise the question of the link between histone methylation and acetylation.
Project description:Plant regeneration via somatic embryogenesis is time-consuming and highly genotype-dependent. The plant somatic embryogenesis process provokes many epigenetics changes including DNA methylation and histone modification. Recently, an elite cotton Jin668, with an extremely high regeneration ability, was developed from its maternal inbred Y668 cultivar using a Successive Regeneration Acclimation (SRA) strategy. To reveal the underlying mechanism of SRA, we carried out a genome-wide single-base resolution methylation analysis for nonembryogenic calluses (NECs), ECs, somatic embryos (SEs) during the somatic embryogenesis procedure and the leaves of regenerated offspring plants. Jin668 (R4) regenerated plants were CHH hypomethylated compared with the R0 regenerated plants of SRA process. The increase in CHH methylation from NEC to EC was demonstrated to be associated with the RNA-dependent DNA methylation (RdDM) and the H3K9me2-dependent pathway. Intriguingly, the hypomethylated CHH differentially methylated regions (DMRs) of promoter activated some hormone-related and WUSCHEL-related homeobox genes during the somatic embryogenesis process. Inhibiting DNA methylation using zebularine treatment in NEC increased the number of embryos. Our multi-omics data provide new insights into the dynamics of DNA methylation during the plant tissue culture and regenerated offspring plants. This study also reveals that induced hypomethylation (SRA) may facilitate the higher plant regeneration ability and optimize maternal genetic cultivar.
Project description:Methylation of lysine residues, catalyzed by histone methyltransferase (HMT) enzymes, is one of many modifications of core histone proteins that regulate transcription and chromatin structure. G9a is the predominant HMT in mammalian euchromatin and recent data suggest that it is required to perpetuate a malignant phenotype in cancer cells and is implicated in metastasis, supporting this HMT as a therapeutic target for cancer and other diseases associated with epigenetic regulation. Of the methods currently used to measure methyltransferase activity, many involve a separation step or utilize coupling enzymes complicating implementation and data interpretation. Here we describe a homogeneous assay to measure G9a HMT activity using the chemiluminescence-based AlphaScreen immunoassay technology. Methylation of biotinylated-histone peptide is measured through specific antibody-based detection, in conjunction with streptavidin-coated donor and secondary antibody-coated acceptor beads. The method is particularly well suited for detection of inhibitors acting by the desired histone peptide competitive mechanism and is applicable to testing other HMTs, demonstrated here with the G9a homolog EHMT1, also known as GLP.
Project description:SK-UT-1 uterine leiomyosarcomas (Ut-LMS) cells were transduced with a fatty acid synthase (FASN)-containing retroviral vector to recapitulate the "lipogenic phenotype of cancer." Consistent with this model, forced expression of FASN enhanced SK-UT-1 proliferation, migration, and cellular motion. Further investigation showed FASN promotes trimethylation of H3K9 (H3K9me3) and acetylation of H3K27 (H3K27ac) in SK-UT-1 cells. In contrast, siRNA targeting of FASN in high endogenous FASN expressing SK-LMS-1 Ut-LMS cells inhibits trimethylation of H3K9 and acetylation of H3K27. Palmitate, the predominant fatty acid product of FASN, increased H3K9me3, H3K27ac and H3K27me3 detection in SK-UT-1 cells. FASN promoted histone 3 methylation and acetylation through alteration of histone 3-modifying enzymatic activities (HDAC, HDM, HMT and HAT). ChIP-seq in SK-UT-1-FASN cells with anti-H3K9me3 antibody identified regions of enriched binding compared to vector-only cells. One differentially-enriched gene, CRISP1, was investigated further by ChIP-PCR. The transcriptionally repressive function of H3K9me3 was confirmed in CRISP1. Our results provide mechanistic insight into the pathobiology of the "lipogenic phenotype of cancer." Here, FASN reprograms the Ut-LMS epigenome through chromatin remodeling to promote the "malignant phenotype."
Project description:BACKGROUND: The rubber tree (Hevea brasiliensis) is native to the Amazon region and it is the major source of natural rubber in the world. Rubber tree breeding is time-consuming and expensive. However, molecular markers such as microsatellites can reduce the time required for these programs. This study reports new genomic microsatellite markers developed and characterized in H. brasiliensis and the evaluation of their transferability to other Hevea species. FINDINGS: We constructed di- and trinucleotide-enriched libraries. From these two libraries, 153 primer pairs were designed and initially evaluated using 9 genotypes of H. brasiliensis. A total of 119 primer pairs had a good amplification product, 90 of which were polymorphic. We chose 46 of the polymorphic markers and characterized them in 36 genotypes of H. brasiliensis. The expected and observed heterozygosities ranged from 0.1387 to 0.8629 and 0.0909 to 0.9167, respectively. The polymorphism information content (PIC) values ranged from 0.097 to 0.8339, and the mean number of alleles was 6.4 (2-17). These 46 microsatellites were also tested in 6 other Hevea species. The percentage of transferability ranged from 82% to 87%. Locus duplication was found in H. brasiliensis and also in 5 of other species in which transferability was tested. CONCLUSIONS: This study reports new microsatellite markers for H. brasiliensis that can be used for genetic linkage mapping, quantitative trait loci identification and marker- assisted selection. The high percentage of transferability may be useful in the evaluations of genetic variability and to monitor introgression of genetic variability from different Hevea species into breeding programs.
Project description:Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.