Biology subjects

Chansard, A.

Publications and source records attributed to Chansard, A..

3 recordsLinked to original sources

bioRxiv2024

Proteomic profiling of UV damage repair patches uncovers histone chaperones with central functions in chromatin repair

DNA damage compromises not only genome stability but also the integrity of the chromatin template, which plays a central role in controlling cell identity. Our understanding of chromatin repair mechanisms is very incomplete. To bridge this knowledge gap, here we devise a novel proteomic strategy to characterize dynamic changes in the chromatin landscape during the repair of UV-induced DNA lesions in human cells, in a quantitative, unbiased and time-resolved manner. Thus, we identify the histone chaperones DNAJC9 and MCM2 as central players in chromatin repair. We demonstrate that DNAJC9 and MCM2 are independently recruited to sites of UV damage repair. DNAJC9 provides new H3-H4 histones to CAF-1 and HIRA chaperones for deposition into chromatin and also stimulates old H3-H4 histone recovery. DNAJC9 cooperates with MCM2 to coordinate old and new histone dynamics during UV damage repair. Together, our proteomic dataset provides a molecular framework for further dissecting epigenome maintenance mechanisms.

molecular biology↗

bioRxiv2020

Unveiling the inter individual variability of human fibroblasts innate immune response using robust cell-based protocols

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our "Add-only" protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

immunology↗

bioRxiv2019

Histone chaperones and modifiers cooperate to maintain heterochromatin integrity following DNA damage.

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV, SETDB1 coordinating histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

molecular biology↗