Additive inheritance of histone modifications in Arabidopsis thaliana intra-specific hybrids

Plant genomes are earmarked with defined patterns of chromatin marks. Little is known about the stability of these epigenomes when related, but distinct genomes are brought together by intra‐species hybridization. Arabidopsis thaliana accessions and their reciprocal hybrids were used as a model system to investigate the dynamics of histone modification patterns. The genome‐wide distribution of histone modifications H3K4me2 and H3K27me3 in the inbred parental accessions Col‐0, C24 and Cvi and their hybrid offspring was compared by chromatin immunoprecipitation in combination with genome tiling array hybridization. The analysis revealed that, in addition to DNA sequence polymorphisms, chromatin modification variations exist among accessions of A. thaliana. The range of these variations was higher for H3K27me3 (typically a repressive mark) than for H3K4me2 (typically an active mark). H3K4me2 and H3K27me3 were rather stable in response to intra‐species hybridization, with mainly additive inheritance in hybrid offspring. In conclusion, intra‐species hybridization does not result in gross changes to chromatin modifications.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Epigenetics and parental effects

Parental effects are a major source of phenotypic plasticity and may influence offspring phenotype in concert with environmental demands. Studies of “environmental epigenetics” suggest that (1) DNA methylation states are variable and that both demethylation and remethylation occur in post‐mitotic cells, and (2) that remodeling of DNA methylation can occur in response to environmentally driven intracellular signaling pathways. Studies of mother‐offspring interactions in rodents suggest that parental signals influence the DNA methylation, leading to stable changes in gene expression. If parental effects do indeed enhance the “match” between prevailing environmental demands and offspring phenotype, then the potential for variation in environmental conditions over time would suggest a mechanism that requires active maintenance across generations through parental signaling. We suggest that parental regulation of DNA methylation states is thus an ideal candidate mechanism for parental effects on phenotypic variation.     

An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.     

A tag-based approach for high-throughput analysis of CCWGG methylation

Non-CpG methylation occurring in the context of CNG sequences is found in plants at a large number of genomic loci. However, there is still little information available about non-CpG methylation in mammals. Efficient methods that would allow detection of scarcely localized methylated sites in small quantities of DNA are required to elucidate the biological role of non-CpG methylation in both plants and animals. In this study, we tested a new whole genome approach to identify sites of CCWGG methylation (W is A or T), a particular case of CNG methylation, in genomic DNA. This technique is based on digestion of DNAs with methylation-sensitive restriction endonucleases EcoRII-C and AjnI. Short DNAs flanking methylated CCWGG sites (tags) are selectively purified and assembled in tandem arrays of up to nine tags. This allows high-throughput sequencing of tags, identification of flanking regions, and their exact positions in the genome. In this study, we tested specificity and efficiency of the approach.     

Cell-wall pectin and its degree of methylation in the maize root-apex: significance for genotypic differences in aluminium resistance

Cell-wall (CW) pectin content and its degree of methylation in root apices of selected maize cultivars were studied in relation to genotypic Al resistance. Maize cultivars differing in Al resistance were grown in nutrient solution treated with or without Al, and pectin content of the root tips was determined. Control plants did not differ in pectin content in the 5 mm root apex. Al treatment increased the pectin content of the root apex in all cultivars but more prominently in the Al-sensitive cultivars. Pectin and Al contents in 1 mm root sections decreased from the apex to the 3–4 mm zone. Pectin contents of the apical root sections were consistently higher although significantly different only in the 1–2 mm zone in the Al-sensitive cv Lixis. Al contents in most root sections were significantly higher in cv Lixis than in Al-resistant cv ATP-Y. Localization of pectins by immunofluorescence revealed that Al-sensitive cv. Lixis has a higher proportion of low-methylated pectin and thus a higher negativity of the cell wall than Al-resistant cv ATP-Y. This is in agreement with the higher Al content and Al sensitivity of cv Lixis. It is concluded that differences in CW pectin and its degree of methylation contribute to genotypic differences in Al resistance in maize in addition to the release of organic acid anions previously reported.