Different Modes of Action of Genetic and Chemical Downregulation of Histone Deacetylases with Respect to Plant Development and Histone Modifications.

A high degree of developmental plasticity enables plants to adapt to continuous, often unfavorable and unpredictable changes in their environment. At the molecular level, adaptive advantages for plants are primarily provided by epigenetic machinery including DNA methylation, histone modifications, and the activity of noncoding RNA molecules. Using a mass spectrometry-based proteomic approach, we examined the levels of acetylated histone peptide forms in Arabidopsis plants with a loss of function of histone deacetylase 6 (HDA6), and in plants germinated in the presence of HDA inhibitors trichostatin A (TSA) and sodium butyrate (NaB). Our analyses revealed particular lysine sites at histone sequences targeted by the HDA6 enzyme, and by TSA- and NaB-sensitive HDAs. Compared with plants exposed to drugs, more dramatic changes in the overall profiles of histone post-translational modifications were identified in hda6 mutants. However, loss of HDA6 was not sufficient by itself to induce hyperacetylation to the maximum degree, implying complementary activities of other HDAs. In contrast to hda6 mutants that did not exhibit any obvious phenotypic defects, the phenotypes of seedlings exposed to HDA inhibitors were markedly affected, showing that the effect of these drugs on early plant development is not limited to the modulation of histone acetylation levels.

Holocaust history is not reflected in telomere homeostasis in survivors and their offspring.

Telomeres, nucleoprotein structures at the ends of eukaryotic chromosomes, are crucial for the maintenance of genome integrity. While the lengths of telomeres at birth are determined genetically, many factors including environmental and living conditions affect the telomere lengths during a lifespan. In this context, extreme and long-term stress has been shown to negatively impact telomeres and their protective function, with even offspring being influenced by the stress experienced by parents. Using quantitative PCR, the relative lengths of telomeres of survivors of the Holocaust during World War II and two generations of their offspring were analyzed. These data were related to those of control groups, persons of comparable age without a strong life stress experience. In contrast to previous studies of other stress-exposed groups, the relative lengths of telomeres were comparable in groups of persons exposed to Holocaust-related stress and their progenies, and in control groups. Interestingly, shorter telomeres of Holocaust survivors of the age under 12 in the year 1945 compared to Holocaust survivors of the age above 12 were detected. Our results are discussed with respect to certain exceptionality of persons having been able to cope with an extreme stress more than 70 years ago and living to a very old age.

Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones.

Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following chemical derivatization of histone lysine residues. Here, we report a straightforward approach for plant histone sample preparation for mass spectrometry, based on filter-aided sample preparation coupled with histone propionylation. The approach offers savings in sample handling and preparation time, enables removal of interfering compounds from the sample, and does not require either precipitation or dialysis of histone extract. We show the comparison of two protocol variants for derivatization of histone proteins, in-solution propionylation in the vial and propionylation on the filter unit. For both protocols, we obtained identical abundances of post-translationally modified histone peptides. Although shorter time is required for histone protein labeling on the filter unit, in-solution derivatization slightly outweighed filter-based variant by lower data variability. Nevertheless, both protocol variants appear to be efficient and convenient approach for preparation of plant histones for mass spectrometric analysis.

Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors.

Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.