Purpose: Radiation-induced senescence is characterized by profound changes in chromatin
organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNASegments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent
cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-AssociatedSecretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in
establishing radiation-induced senescence. Experimental Design: Primary and genetically-modified
lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation
and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy.
Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and
transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP
factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry.
Results: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq
and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory
regions decisively modulates transcription factor recruitment and inflammatory gene expression,
resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent
H2A.J expression in response to radiation-induced DNA damage, therefore contributing to proinflammatory tissue reactions. Conclusions: The fine-tuned incorporation of H2A.J defines the
epigenetic landscape for driving the senescence programme in response to radiation-induced DNA
damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into
DNA-damage triggered epigenetic mechanisms governing the biological processes of radiationinduced injury