Relationship between DNA methylation and histone acetylation levels, cell redox and cell differentiation states in sugarbeet lines

Abstract

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.

DNA methylation and histone acetylation: genotypic variations in hybrid poplars, impact of water deficit and relationships with productivity

“…Using polyclonal antibodies raised against acetylated lysine, we showed that acetylation ratios of histones H 3 and H 4 were consistent with previously-reported values (Causevic et al, 2006). No significant difference could be observed between genotypes or in response to a moderate water deficit.…”

“…Acetylation ratios of histones H 3 or H 4 were calculated according to the following formula: acetylation ratio of H 3 or H 4 histone (AcH 3 or AcH 4 ) = amount of acetylated histone H 3 or H 4 / amount of non acetylated histone H 3 . Anti-acetylated histone H 3 antibodies were also tested to confirm the data as previously published (Causevic et al, 2006).…”

“…Nevertheless, a detailed kinetic study of the variation of histone acetylation ratios on various lysine positions during water deficit as well as a gene specific analysis are needed to reach firm conclusions. Several HDAC isoforms were assessed in hybrid poplars in accordance with poplar databases (Tuskan et al, 2006) and a limited proteolysis of the C-terminal part previously reported in other plants (Causevic et al, 2006;Loidl, 2004;Pipal et al, 2003). The relative amount of a 80 kDa HDAC isoform was positively correlated to the total HDAC enzymatic activity.…”

“…Total soluble proteins and chromatin bound histones were extracted separately from shoot apex according to Causevic et al (2006). The protein concentration was determined using Protein Assay reagent (Bio-Rad, Marnes-la-Coquette, France) and a standard curve established with different solutions of bovine serum albumin (from 0 to 33 μg μL −1 ).…”

“…Blotted membranes with soluble proteins were hybridized with different antibodies: carrot anti-DNA methyltransferase 1 (DNMT1; Bernacchia et al, 1998;Causevic et al, 2005) antibodies at 1:300 dilution (v/v) and three maize anti-Histone deacetylase antibodies (HDAC; Pipal et al, 2003;Causevic et al, 2006) raised against the three classes of HDAC (anti-HD1A at 1:2000 dilution (v/v), anti-HD1BI at 1:1500 dilution (v/v) and anti-HD2 at 1:1000 dilution (v/v)). Blotted membranes with histone extracts were hybridized with anti-acetylated lysine at 1:1000 dilution (v/v) or anti-non acetylated H 3 histone at 1:200 dilution (v/v) (Santa Cruz Biotechnology, California, and Cell Signalling Technology, Danvers, USA).…”