UV-B-induced modulation of constitutive heterochromatin content in Arabidopsis thaliana.

Sunlight regulates transcriptional programs and triggers the shaping of the genome throughout plant development. Among the different sunlight wavelengths that reach the surface of the Earth, UV-B (280-315 nm) controls the expression of hundreds of genes for the photomorphogenic responses and also induces the formation of photodamage that interfere with genome integrity and transcriptional programs. The combination of cytogenetics and deep-learning-based analyses allowed determining the location of UV-B-induced photoproducts and quantifying the effects of UV-B irradiation on constitutive heterochromatin content in different Arabidopsis natural variants acclimated to various UV-B regimes. We identified that UV-B-induced photolesions are enriched within chromocenters. Furthermore, we uncovered that UV-B irradiation promotes constitutive heterochromatin dynamics that differs among the Arabidopsis ecotypes having divergent heterochromatin contents. Finally, we identified that the proper restoration of the chromocenter shape, upon DNA repair, relies on the UV-B photoreceptor, UV RESISTANCE LOCUS 8 (UVR8). These findings shed the light on the effect of UV-B exposure and perception in the modulation of constitutive heterochromatin content in Arabidopsis thaliana.

Advanced Image Analysis Methods for Automated Segmentation of Subnuclear Chromatin Domains.

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated (iCRAQ) and one based on deep learning (Nucl.Eye.D), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics.

Formation and Recognition of UV-Induced DNA Damage within Genome Complexity.

Ultraviolet (UV) light is a natural genotoxic agent leading to the formation of photolesions endangering the genomic integrity and thereby the survival of living organisms. To prevent the mutagenetic effect of UV, several specific DNA repair mechanisms are mobilized to accurately maintain genome integrity at photodamaged sites within the complexity of genome structures. However, a fundamental gap remains to be filled in the identification and characterization of factors at the nexus of UV-induced DNA damage, DNA repair, and epigenetics. This review brings together the impact of the epigenomic context on the susceptibility of genomic regions to form photodamage and focuses on the mechanisms of photolesions recognition through the different DNA repair pathways.

Photodamage repair pathways contribute to the accurate maintenance of the DNA methylome landscape upon UV exposure.

Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photodamaged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromocenter reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photolesions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair.