Gabriela Lochmanová

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Post-Translational Modifications of Histones in Human Sperm

We examined the levels and distribution of post‐translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM‐FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM‐FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non‐integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC‐MS/MS and MALDI‐TOF MS analysis. Taken together, these data indicate that particular post‐translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. J. Cell. Biochem. 116: 2195–2209, 2015.

A combined approach for the study of histone deacetylase inhibitors

Overexpression of histone deacetylases (HDACs), with consequent hypoacetylation of histones, is reportedly associated with transcriptional repression of tumour suppressor genes. Thus, inhibition of HDACs has emerged as a promising strategy in cancer therapy. In order to monitor the effects of potential HDAC inhibitors, a multi-level approach consisting of preliminary screening (measurement of HDAC activity and semi-quantitative evaluation of histone H4 modification profile by MALDI-TOF MS) and detailed analysis of histone modification forms (using 2-D AUT/AU PAGE and LC-ESI-IT MS) has been used in this study. The data obtained provide a global insight into the effects of HDAC inhibitors on the histone acetylation status that participates in gene transcription control. Using two example inhibitors, valproic acid sodium salt and entinostat, we show that similar levels of HDAC inhibition induced by different agents can lead to distinct rates of histone hyperacetylation, suggesting that except for the direct inhibition of HDACs, additional molecular mechanisms amplifying the response are likely to be involved in the inhibitory process. The approach used in our study makes it possible not only to follow the dynamics of individual histone modification forms, but also of their combined occurrence in the N-terminal fragment.

Cell differentiation along multiple pathways accompanied by changes in histone acetylation status

Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.

Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.

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.

Preventing the mixed detergent micelle effect in two-dimensional electrophoresis on Tris-Tricine gels

Application of Tris‐N‐[Tris(hydroxymethyl)methyl]glycine gels for 2DE is hampered by formation of mixed CHAPS–SDS micelles resulting in typical swirling pattern in the low mass range, which makes reliable quantitative and qualitative gel evaluation impossible. Modification of 2DE strip equilibration procedure prevented the direct interaction between both detergents during equilibration process, thus substantially improving gel separation.

Potential biomarkers for early detection of acute graft-versus-host disease

Acute graft‐versus‐host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HCT), resulting in considerable morbidity and mortality. Currently, the diagnosis of aGVHD is largely made based on clinical parameters and invasive biopsies. For the past 20 years, researchers have been trying to find reliable biomarkers to enable early and accurate diagnosis of aGVHD. Although a number of potential aGVHD biomarkers have been published, as yet, no validated diagnostic test is available. Proteomics encompasses a broad range of rapidly developing technologies, which have shown tremendous promise for early detection of aGVHD. In this article, we review the current state of aGVHD biomarker discovery, provide a summary of the key proteins of interest and the most common analytical procedures for the clinic, as well as outlining the significant challenges faced in their use.

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.

Irradiation by γ-rays reduces the level of H3S10 phosphorylation and weakens the G2 phase-dependent interaction between H3S10 phosphorylation and γH2AX

Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX.