Functional analysis of chromatin-associated proteins inSordaria macrosporareveals similar roles for RTT109 and ASF1 in development and DNA damage response

Abstract

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomyceteSordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase ofNeurospora crassathat performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a significant decrease in H3K56 acetylation levels, as detected by semiquantitative western blot analysis. Deletion mutants ofrtt109andasf1are sensitive to the DNA damaging agent MMS (methylmethane sulfonate), but not HU (hydroxyurea). In contrast,chk2mutants are fertile and resistant to MMS, but not HU. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.