Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals 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 ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair 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 of Neurospora crassa that 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 loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. 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.

Keywords: Sordaria macrospora; asf1; chk2; rtt109; DNA damage response; chromatin-associated proteins; fruiting body development; functional genomics.

Fig. 1.

Development of rtt109 deletion mutants compared to wild-type and complementation strains. S. macrospora strains lacking the rtt109 gene were found to exhibit impaired development with a block at the stage of early protoperithecia formation. Upon reintroduction of rtt109, normal life cycle progression was fully restored, as evidenced by the formation of late, melanized protoperithecia, and the development of fully formed perithecia indistinguishable from those of the wild-type strain. The scale bar for ascogonia and protoperithecia represents 20 µm, while the scale bar for perithecia represents 100 µm.

Fig. 2.

Comparison of vegetative growth of rtt109 deletion and complementation strains with wild-type and asf1 deletion strains. Mycelial spread was observed over a period of 96 h and a final time after 10 days. While the wild-type almost covered the entire plate in 96 h, S. macrospora Δrtt109 was significantly slower and grew as slowly as Δasf1 strains. This effect was reversed by reintroduction of rtt109 in the rtt109 deletion mutant, complementing the growth defect of the mutant. A visible difference between Δrtt109 and Δasf1 was the density of the mycelium. S. macrospora Δrtt109 appeared to grow as densely as the wild-type, while Δasf1 appeared to be thinner overall. Scale bar represent 1 cm.

Fig. 3.

Quantification of the vegetative growth rates of S. macrospora Δrtt109, the respective complementation strain, Δasf1 and the wild-type. While the wild-type grew at about 2.5 cm per day, deletion of rtt109 or asf1 resulted in a significant decrease in growth rate to about 1.5 cm per day. Complementation of rtt109 mutants restored the growth rate to wild-type levels. Quantification was performed for five independent replicates and significance was assessed by Student's t-test. ***P-value < 0.001, n.s. = P-value > 0.05.

Fig. 4.

MMS sensitivity test for S. macrospora Δrtt109. The sensitivity of S. macrospora rtt109 deletion mutants to the genotoxic compound MMS was observed after 4 days of growth on BMM media and BMM media supplemented with 0.007% MMS. The addition of MMS completely halted the growth of the rtt109 and asf1 deletion mutants. When rtt109 was reintroduced into Δrtt109, its ability to survive under MMS stress was restored. The scale bar provided represents 1 cm.

Fig. 5.

HU sensitivity test for S. macrospora Δrtt109. After a 4-day incubation on BMM media supplemented with 8 mM HU, the sensitivity of rtt109 deletion mutants was assessed. S. macrospora Δrtt109 exhibited no visible sensitivity to this DNA damaging agent, resembling the resistance observed in Δasf1. Scale bar represents 1 cm.

Fig. 6.

Localization analysis of RTT109 by fluorescence microscopy. RTT109 was expressed as a fusion protein with an eGFP tag and cultivated together with a strain expressing histone H3 fused to an mRFP tag. The visible colocalization of the eGFP and mRFP (dsRed) fluorescence indicates a nuclear localization of RTT109. No eGFP autofluorescence was detectable in the reference strain expressing only H2A-mRFP. Scale bar represents 20 µm.

Fig. 7.

Assessment of H3K56ac levels in S. macrospora Δrtt109. The level of global H3K56ac was determined by comparing equal amounts of whole protein extracts from the wild-type, Δrtt109, and the respective complementation strain by SDS-PAGE separation and western blotting with H3K56ac-specific antibodies. H3 antibodies were used to assess equal loading and comparable amounts of histone 3 in the protein extracts. In three biological replicates, no signal for H3K56ac was detectable in Δrtt109 strains. This effect was complemented by reintroduction of rtt109 in the deletion mutant. Uncropped blots and the corresponding Coomassie gels are shown in Supplementary Fig. 3.

Fig. 8.

Growth comparison of S. macrospora Δchk2 with Δasf1 and the wild-type. The growth and overall characteristics of the strains were monitored for 96 h, with a final observation after 7 days on BMM media. There were no noticeable differences between the chk2 deletion mutant and the wild-type strain. Both strains exhibited similar growth rates and developed visible perithecia. The Δasf1 strain displayed significantly slower growth and failed to produce perithecia throughout the observation period. White dashed circles indicate the growth front. Scale bar represents 1 cm.

Fig. 9.

Morphological comparison of sexual structures generated by S. macrospora Δchk2 with those of the wild-type. The chk2 deletion mutant demonstrated the ability to undergo the complete life cycle of S. macrospora without any impairments. The fruiting bodies produced by the mutant exhibited no abnormalities in comparison to the wild-type strain. The perithecia were formed and positioned in a normal manner and the overall morphology of the perithecia did not display any visible defects. Δchk2 strains generated wild-type-like asci. Scale bars represent 1 mm for top-down view, 500 µm for side view, and 100 µm for asci.

Fig. 10.

MMS sensitivity test for S. macrospora Δchk2. The growth of the strains was monitored on BMM media supplemented with 0.007% MMS for a duration of 96 h. The S. macrospora Δchk2 strain did not display increased sensitivity to the DNA damaging agent compared to the wild-type strain. Consistent with previous observations, the asf1 deletion mutant exhibited a high level of sensitivity to MMS. White dashed circles indicate the growth front. Scale bar represents 1 cm.

Fig. 11.

HU sensitivity test for S. macrospora Δchk2. The growth of the strains was observed on BMM media supplemented with 8 mM HU for a period of 96 h. In contrast to the observations with MMS, the chk2 deletion mutant exhibited sensitivity to the DNA damaging agent HU. While Δchk2 strains appeared to progress normally for the first 48 h, they stopped growing after that time. Reintroduction of the chk2 gene restored HU resistance. In contrast, the wild-type and Δasf1 strains continued to grow under the same conditions. White dashed circles indicate the growth front. Scale bar represents 1 cm.