Gabriela Lochmanová

Central European Institute of Technology – Masaryk University

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Zbyněk Zdráhal

Central European Institute of Technology

Soňa Legartová

Czech Academy of Sciences, Institute of Biophysics

Eva Bártová

Czech Academy of Sciences

Miloslava Fojtová

Central European Institute of Technology

Stanislav Kozubek

Czech Academy of Sciences, Institute of Biophysics

Jiřı́ Fajkus

Central European Institute of Technology

Hana Kuchaříková

Central European Institute of Technology

Hana Konečná

Nemocnice České Budějovice

Sylva Brabencová

Masaryk University

David Potěšil

Central European Institute of Technology

Cooperative Institutions

Masaryk University

Central European Institute of Technology

Central European Institute of Technology – Masaryk University

Czech Academy of Sciences, Institute of Biophysics

Czech Academy of Sciences

University Hospital Brno

Mendel University in Brno

Palacký University Olomouc

Medical University of Vienna

St. Anne's University Hospital Brno

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

Frontiers in Plant Science (2017)

Sylva Brabencová Ivana Ihnatová David Potěšil Miloslava Fojtová Jiřı́ Fajkus

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.

Ecotype

10.3389/fpls.2017.02084

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

Biopolymers and Cell (2019)

Eva Bártová Gabriela Lochmanová Soňa Legartová Jana Suchánková Radek Fedr

10.7124/bc.0009eb

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Citations (0)

Quantitative Analysis of Posttranslational Modifications of Plant Histones

Methods in molecular biology (2022)

Hana Kuchaříková Zuzana Plšková Zbyněk Zdráhal Miloslava Fojtová Pavel Kerchev

Histone-modifying enzymes

10.1007/978-1-0716-2469-2_18

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Post‐Translational Modifications of Histones in Human Sperm

Journal of Cellular Biochemistry (2015)

Jana Krejčí Lenka Stixová Eva Pagáčová Soňa Legartová Stanislav Kozubek

ABSTRACT 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. © 2015 Wiley Periodicals, Inc.

Histone H4

10.1002/jcb.25170

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Disruption ofNAP1genes inArabidopsis thalianasuppresses thefas1mutant phenotype, enhances genome stability and changes chromatin compaction

The Plant Journal (2020)

Karolína Kolářová Martina Nešpor Dadejová Tomáš Loja Gabriela Lochmanová Eva Sýkorová

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.

Histone code

10.1111/tpj.15145

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

Journal of Experimental Botany (2008)

Gabriela Lochmanová Zbyněk Zdráhal Hana Konečná Šárka Koukalová Jiří Malbeck

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.

Photomorphogenesis

Etiolation

Cotyledon

10.1093/jxb/ern220

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Trimethylacetic Anhydride–Based Derivatization Facilitates Quantification of Histone Marks at the MS1 Level

Molecular & Cellular Proteomics (2021)

Hana Kuchaříková Pavlína Dobrovolná Gabriela Lochmanová Zbyněk Zdráhal

Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom–up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.

10.1016/j.mcpro.2021.100114

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

Frontiers in Plant Science (2018)

Dominika Ledvinová Kamil Mikulášek Hana Kuchaříková Sylva Brabencová Miloslava Fojtová

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.

Sample Preparation

10.3389/fpls.2018.01373

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

Biochemistry and Cell Biology (2014)

Soňa Legartová Stanislav Kozubek Michal Franek Zbyněk Zdráhal Gabriela Lochmanová

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.

Sodium butyrate

Histone H4

HDAC4

SAP30

HDAC11

10.1139/bcb-2013-0082

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Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin

Biochimie (2012)

Soňa Legartová Alžběta Jugová Lenka Stixová Stanislav Kozubek Miloslava Fojtová

Demethylase

Histone Methylation

10.1016/j.biochi.2012.09.027

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Citations (10)