Tissue-Specific Epigenetic Modifications in Root Apical Meristem Cells of Hordeum vulgare

Epigenetic modifications of chromatin structure are essential for many biological processes, including growth and reproduction. Patterns of DNA and histone modifications have recently been widely studied in many plant species, although there is virtually no data on the spatial and temporal distribution of epigenetic markers during plant development. Accordingly, we have used immunostaining techniques to investigate epigenetic modifications in the root apical meristem of Hordeum vulgare. Histone H4 acetylation (H4K5ac), histone H3 dimethylation (H3K4me2, H3K9me2) and DNA methylation (5mC) patterns were established for various root meristem tissues. Distinct levels of those modifications were visualised in the root cap, epidermis, cortex and vascular tissues. The lateral root cap cells seem to display the highest level of H3K9me2 and 5mC. In the epidermis, the highest level of 5mC and H3K9me2 was detected in the nuclei from the boundary of the proximal meristem and the elongation zone, while the vascular tissues were characterized by the highest level of H4K5ac. Some of the modified histones were also detectable in the cytoplasm in a highly tissue-specific manner. Immunolocalisation of epigenetic modifications of chromatin carried out in this way, on longitudinal or transverse sections, provides a unique topographic context within the organ, and will provide some answers to the significant biological question of tissue differentiation processes during root development in a monocotyledon plant species.     

Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

Identification and Characterization of an Epi-Allele of FIE1 Reveals a Regulatory Linkage between Two Epigenetic Marks in Rice

DNA methylation and histone H3 Lys 9 dimethylation (H3K9me2) are important epigenetic repression marks for silencing transposons in heterochromatin and for regulating gene expression. However, the mechanistic relationship to other repressive marks, such as histone H3 Lys 27 trimethylation (H3K27me3) is unclear. FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1) encodes an Esc-like core component of the Polycomb repressive complex 2, which is involved in H3K27me3-mediated gene repression. Here, we identify a gain-of-function epi-allele (Epi-df) of rice (Oryza sativa) FIE1; this allele causes a dwarf stature and various floral defects that are inherited in a dominant fashion. We found that Epi-df has no changes in nucleotide sequence but is hypomethylated in the 5′ region of FIE1 and has reduced H3K9me2 and increased H3K4me3. In Epi-df, FIE1 was ectopically expressed and its imprinting was disrupted. FIE1 interacted with rice Enhancer of Zeste homologs, consistent with its role in H3K27me3 repression. Ectopic expression of FIE1 in Epi-df resulted in alteration of H3K27me3 levels in hundreds of genes. In summary, this work identifies an epi-allele involved in H3K27me3-mediated gene repression that itself is highly regulated by DNA methylation and histone H3K9me2, thereby shedding light on the link between DNA methylation and histone methylation, the two important epigenetic marks regulating rice development.     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.     

New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Dot1-Dependent Histone H3K79 Methylation Promotes the Formation of Meiotic Double-Strand Breaks in the Absence of Histone H3K4 Methylation in Budding Yeast

Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.     

Comparative analysis of Histone modifications and DNA methylation at <Emphasis Type="Italic">OsBZ8</Emphasis> locus under salinity stress in IR64 and Nonabokra rice varieties

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region ?2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5′ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the A^vy LTR and exhibit constitutive ectopic expression of Agouti (a), also display enrichment of H3 and H4 di-acetylation (p = 0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p = 0.01). No differences are observed for H3K4 tri-methylation (p = 0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.Key words: epigenetics, metastable epiallele, viable yellow agouti, histone, environmental epigenomics