Cristel C Carles | PubMed Health

PubMed2025

dCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.

Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.

Chromatin↗

PubMed2025

Quantitative Profiling of Histone Variants and Posttranslational Modifications by Tandem Mass Spectrometry in Arabidopsis.

Histone dynamics constitute an important layer of gene regulations associated with development and growth in multicellular eukaryotes. They also stand as key determinants of plant responses to environmental changes. Histone dynamics include the exchange of histone variants as well as post-translational modifications of their amino acid residues (such as acetylation and mono/di/trimethylation), commonly referred to as histone marks. Investigating histone dynamics with a focus on combinatorial changes occurring at their residues will greatly help unravel how plants achieve phenotypic plasticity.Mass spectrometry (MS) analysis offers unequaled resolution of the abundance of histone variants and of their marks. Indeed, relative to other techniques such as western blot or genome-wide profiling, this powerful technique allows quantifying the relative abundances of histone forms, as well as revealing coexisting marks on the same histone molecule. Yet, while MS-based histone analysis has proven efficient in several animals and other model organisms, this method stands out as more challenging in plants. One major challenge is the isolation of sufficient amounts of pure, high-quality histones, likely rendered difficult by the presence of the cell wall, for sufficiently deep and resolutive identification of histone species.In this chapter, we describe a straightforward MS-based proteomic method, implemented to characterize histone marks from Arabidopsis thaliana seedling tissues and cell culture suspensions. After acid extraction of histones, in vitro propionylation of free lysine residues, and digestion with trypsin, a treatment at highly basic pH allows obtaining sharp spectral signals of biologically relevant histone peptide forms.The method workflow described here shall be used to measure changes in histone marks between Arabidopsis thaliana genotypes, along developmental time-courses, or upon various stresses and treatments.

Histones↗