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1.
New Phytol ; 243(3): 966-980, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38840557

ABSTRACT

Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Cycle , DNA Damage , Gene Expression Regulation, Plant , Plant Roots , Rad51 Recombinase , Transcription, Genetic , Arabidopsis/genetics , Plant Roots/genetics , Plant Roots/cytology , Cell Cycle/genetics , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism
2.
Mol Cell ; 84(12): 2255-2271.e9, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38851186

ABSTRACT

The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Gene Expression Regulation, Plant , Gene Silencing , Histones , MADS Domain Proteins , Polycomb Repressive Complex 2 , RNA Polymerase II , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Histones/metabolism , Histones/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Polycomb Repressive Complex 2/metabolism , Polycomb Repressive Complex 2/genetics , MADS Domain Proteins/genetics , MADS Domain Proteins/metabolism , Transcription, Genetic , Polyadenylation , Histone Demethylases/metabolism , Histone Demethylases/genetics , Transcription Termination, Genetic , Chromatin/metabolism , Chromatin/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics
3.
Elife ; 122023 07 19.
Article in English | MEDLINE | ID: mdl-37466633

ABSTRACT

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Epigenesis, Genetic , Gene Silencing , MADS Domain Proteins/genetics , MADS Domain Proteins/metabolism , Gene Expression , Gene Expression Regulation, Plant , Flowers/physiology , Cold Temperature
4.
EMBO Rep ; 23(12): e54736, 2022 12 06.
Article in English | MEDLINE | ID: mdl-36278395

ABSTRACT

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double-strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP-tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1-1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.


Subject(s)
Chromatin , DNA Damage , Chromatin/genetics
5.
Elife ; 102021 09 02.
Article in English | MEDLINE | ID: mdl-34473050

ABSTRACT

The histone modification H3K27me3 plays a central role in Polycomb-mediated epigenetic silencing. H3K27me3 recruits and allosterically activates Polycomb Repressive Complex 2 (PRC2), which adds this modification to nearby histones, providing a read/write mechanism for inheritance through DNA replication. However, for some PRC2 targets, a purely histone-based system for epigenetic inheritance may be insufficient. We address this issue at the Polycomb target FLOWERING LOCUS C (FLC) in Arabidopsis thaliana, as a narrow nucleation region of only ~three nucleosomes within FLC mediates epigenetic state switching and subsequent memory over many cell cycles. To explain the memory's unexpected persistence, we introduce a mathematical model incorporating extra protein memory storage elements with positive feedback that persist at the locus through DNA replication, in addition to histone modifications. Our hybrid model explains many features of epigenetic switching/memory at FLC and encapsulates generic mechanisms that may be widely applicable.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation, Plant , Polycomb Repressive Complex 2/genetics , Arabidopsis/physiology , DNA Replication , Histones/metabolism , Protein Processing, Post-Translational
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