Alterations in Linker Histone Dynamics upon DNA Damage

Abstract

Cells are continuously exposed to DNA damaging agents and have evolved numerous pathways to recognize and repair damage. Defects in the DNA damage response (DDR) pathways have been implicated in many human diseases, including cancers, and there are a handful of approved therapeutic drugs that target these pathways. Although DNA repair has been extensively studied, the initiation of these pathways, specifically the ability to sense DNA damage, remains elusive. In vivo, genomic DNA exists in the context of chromatin, and existing evidence suggests that chromatin proteins are directly involved in the DDR. We hypothesize that H1 linker histones play an important role in the recognition and initiation of the single‐strand break repair pathway. H1 binds to DNA between nucleosomes in a dynamic manner, continuously exchanging positions, with an average chromatin residence time of approximately one minute. This behavior is thought to mediate localized chromatin relaxation to allow transient access to DNA‐binding proteins involved in transcription, replication, or repair. To determine if DNA damage has a direct effect on H1 dynamics, we generated a stable murine fibroblast cell line expressing a photo‐convertible fluorescently tagged H1 (pSMOrange‐H1o) and measured H1 dynamics via live cell fluorescence microscopy. We observed that H1 was specifically depleted from chromatin in the vicinity of localized DNA damage induced by micro‐irradiation and that H1 from non‐irradiated chromatin was unable to enter the damaged region for at least ten minutes. By inducing damage and photoconverting H1 in the same region, we found that the off‐rate of H1 from chromatin in the vicinity of damaged DNA was significantly faster (p<0.001) than the off‐rate from chromatin in the vicinity of undamaged DNA. We further noted that the eviction of H1 from chromatin containing damaged DNA was temporally correlated with the recruitment of the early responding DDR protein, poly(ADP‐ribose) polymerase 1 (PARP1). There is evidence that PARP1 and H1 compete for chromatin binding sites and that H1 may be a target of poly ADP‐ribosylation by PARP1 upon DNA damage. By successive additions of C‐terminal DNA‐binding subdomains of H1, we created vectors capable of expressing mutant H1s with increased DNA binding affinity. These vectors were introduced into cell lines expressing GFP‐tagged PARP1. We observed that both the rate and extent of PARP1 recruitment to chromatin containing damaged DNA was significantly decreased upon the overexpression of mutant H1s with enhanced DNA‐binding affinity. Our results show that H1 dynamics are altered upon DNA damage, resulting in the active evcition and subsequent exclusion from chromatin containing damaged DNA. We also suggest that interactions between the linker histones and PARP1 may be an integral aspect of the DNA damage response.Support or Funding InformationNational Science Foundation, Department of Cell and Molecular Biology, School of Graduate Studies in the Health Sciences, SURE UMMCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.