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.


Introduction
The fruiting bodies of ascomycetes are some of the most intricate structures of the fungal kingdom.While fruiting body formation represents a significant developmental process in the fungal life cycle, our understanding of the specific factors that regulate this differentiation remains limited.Sordaria macrospora, a homothallic ascomycete, has proven to be an excellent model to gain insight into the genetic background of sexual development and multicellular development in general (Teichert et al. 2020).S. macrospora offers multiple advantages as a model organism, such as a very fast life cycle as it generates its complex fruiting bodies, the perithecia, in under 7 days under laboratory conditions (Lord and Read 2011).Multiple approaches, from the investigation of mutants from random mutagenesis programs (Kück et al. 2009) to transcriptomicsbased reverse genetics (Nowrousian 2018), have been used to study the complex genetic network of perithecia formation.Such research has yielded a lot of information about developmental genes, some of which are known to be conserved in higher eukaryotes and are in cases like the components of the STRIPAK complex (Blank-Landeshammer et al. 2019) or the histone chaperone ASF1 (Gesing et al. 2012;Yin et al. 2022) even relevant for human diseases.ASF1 is known to interact with histones H3 and H4, both individually and as a dimer.It is involved in histone assembly and disassembly and was the first histone chaperone discovered to be involved in these processes during DNA replication, repair, and transcription and was long thought to be the only one involved in all three processes (English et al. 2006).While ASF1 facilitates histone transfer and binds non-DNA bound H3-H4, it is not incorporated into the nucleosomes (De Koning et al. 2007).During sexual development of S. macrospora, ASF1 has been shown to be essential for achieving fertility, and Δasf1 mutants arrest their life cycle at the protoperithecia stage (Gesing et al. 2012).Effects on DNA methylation (Schumacher et al. 2018) and histone modification (Breuer et al. 2024) have also been described in S. macrospora asf1 deletion mutants.Since the structure of ASF1 is well characterized and no domains have been detected that would allow it to carry out such enzymatic reactions by itself (English et al. 2006), functions in DNA methylation and histone modification are likely to be carried out by interaction partners.Studies in S. cerevisae showed a loss of H3K56ac histone acetylation in asf1 deletion mutants, and the enzyme identified as responsible for this modification was shown to be the HAT (histone acetyltransferase) Rtt109 (Cote et al. 2019).Indeed, an interaction between Asf1, Rtt109, Vps75 (another histone chaperone) and the target histone H3 has been demonstrated in yeast, and it seems likely that acetylation occurs during the interaction of these proteins (Lercher et al. 2018).Therefore, the loss of H3K56ac in S. cerevisae Δasf1 strains appears to be caused by disruption of the complex that allows Rtt109 to act on its target.The dependence of Rtt109 on interaction partners is one of its distinguishing features, as in vitro experiments with the Saccharomyces cerevisiae proteins have shown that the HAT alone is unable to acetylate histones and only functions in the presence of at least one of its interaction partners Asf1 or Vps75, while the presence of both is required for full activity (Cote et al. 2019).Rtt109 is exclusively found in fungi (Tsubota et al. 2007) and is responsible for modifying specific sites on histone H3, namely K9, K27, and K56, as clearly demonstrated in yeast (Han, Zhou, Horazdovsky et al. 2007;Tang et al. 2011).In addition to its role in histone acetylation, Rtt109 has been shown to be involved in a variety of processes in fungal model organisms.In Fusarium graminearum, it has been shown to be involved in perithecia morphogenesis, ascospore formation, conidiation, and host plant infection (Kong et al. 2018).In Aspergillus flavus it is important for aflatoxin synthesis, virulence, and growth (Sun et al. 2021), whereas Aspergillus fumigatus requires it for normal development and DNA damage response, as well as virulence (Zhang et al. 2021).In Neurospora crassa, a close relative of S. macrospora, RTT109 has been shown to be necessary for the production of small RNAs (Zhang et al. 2014).Therefore, RTT109 can be considered a major factor in many fungal models, suggesting its importance in fungi in general, while the fact that is only found in this group of organisms might make it an interesting target for antifungal drugs.
The histone acetylation generated by RTT109 is closely associated with newly synthesized histones and plays a crucial role in the assembly of nucleosomes during DNA replication and repair processes (Han, Zhou, Horazdovsky et al. 2007).The involvement of H3K56ac in DNA damage repair, and thus the dependence of this process on Rtt109, has been demonstrated in S. cerevisae rtt109 deletion mutants, which lack H3K56ac and are sensitive to the DNA double-strand break inducer methyl methanesulfonate (MMS) and the DNA replication inhibitor hydroxyurea (HU) (Driscoll et al. 2007).A similar relationship between ASF1, RTT109, and H3K56ac might exist in S. macrospora, since asf1 deletion strains of this fungus show sensitivity to MMS and a reduction in H3K56ac (Breuer et al. 2024).Therefore, we hypothesized that RTT109 might be the link between ASF1, histone acetylation, and DNA damage repair in S. macrospora and chose SMAC_05078, the S. macrospora RTT109 homologue, as a target for functional characterization.
Another potential candidate, which might contribute to the reduced genomic stability of asf1 mutants, could be the checkpoint kinase Rad53.Rad53 was shown to be important for DNA damage protection under the influence of MMS and HU in S. cerevisiae (McClure and Diffley 2021).S. cerevisiae Rad53 is known to physically and functionally interact with Asf1, and the activity of Rad53 is tightly regulated by its phosphorylation state.Hypophosphorylated Rad53 is known to be bound to Asf1 and maintained in an inactive state, while phosphorylation and release from the complex is a sign of active Rad53 (Emili et al. 2001).Deletion of asf1 in budding yeast causes defects in DNA damage recovery and may be due to insufficient inactivation of Rad53 (Tsabar et al. 2016).In addition, asf1 deletion mutants show similar sensitivities to MMS and HU as rad53 deletion mutants (Emili et al. 2001).Rad53 is the functional equivalent of checkpoint kinases Chk2 in humans and PRD-4 in the filamentous fungus N. crassa, since the corresponding genes can complement an S. cerevisiae Δrad53 mutant (Matsuoka et al. 1998;Pregueiro et al. 2006).The S. macrospora ortholog to PRD-4/Chk2 is SMAC_00634 (called CHK2 hereafter), which was chosen for analysis to shed more light on the chromatin modifier network that contributes to the DNA damage protection functions of ASF1 in S. macrospora.
In this work, we created deletion mutants of rtt109 and chk2, and analyzed their vegetative growth and potential for sexual development.We also performed genotoxic stress assays to check for similarities to asf1 mutants.Furthermore, we determined the subcellular localization of the RTT109 protein by fluorescence microscopy and estimated the global amount of H3K56Ac in Δrtt109 by western blot analysis.We discuss our findings in the context of the roles of chromatin modifiers during DNA damage protection and regulation of complex multicellular development.

Strains, crosses, and growth conditions
Strains utilized in this study can be found in Supplementary Table 1.These strains were cultivated at a temperature of 25 °C on either solid or liquid cornmeal medium (BMM) or complete medium (CM), following previously established protocols (Esser 1982;Walz and Kück 1995).In order to facilitate genetic crosses, the spore color mutant fus was employed as a partner strain, enabling the identification of recombinant asci (Kück et al. 2009).Previously described methods were used to carry out the transformation of S. macrospora (Walz and Kück 1995).

Cloning procedures, oligonucleotides, and plasmids
The oligonucleotides required for generating plasmids and conducting integration tests have been provided in Supplementary Table 2. Additionally, all the plasmids utilized in this study are listed in Supplementary Table 3.The plasmid containing the deletion sequence for rtt109 was generated through yeast recombinant cloning (Oldenburg et al. 1997).For assembling the deletion plasmid for chk2, and the complementation plasmids for rtt109 and chk2, Golden Gate cloning methodology was employed (Terfrüchte et al. 2014;Dahlmann et al. 2021).

Generation of rtt109 and SMAC_00634 deletion mutants and complementation strains
For conducting gene deletions, homologous recombination was utilized following established procedures (Pöggeler and Kück 2006).In brief, a deletion cassette was constructed, comprising a selection marker in the form of a hygromycin phosphotransferase gene under control of the constitutive Pgpd promoter and TtrpC terminator from Aspergillus nidulans (Punt et al. 1990), flanked by upstream and downstream regions of the target gene.This cassette was then cloned into a vector and transformed into the S. macrospora Δku70 strain after restriction digest (with EcoRI for the rtt106 deletion vector and BamHI for the chk2 deletion vector) and gel elution.Since KU70 is necessary for the ectopic integration of DNA, using a ku70 defective strain facilitates homologous recombination (Pöggeler and Kück 2006).Transformants that exhibited resistance to hygromycin were selected and subsequent crosses were performed with the spore color mutant fus, since most S. macrospora transformants are heterokaryotic.Ascospores from such crosses were isolated to obtain homokaryotic deletion mutants and to enable the elimination of the Δku70 background.The resulting strains were verified through PCR.To complement the rtt109 and chk2 mutants, the assembled plasmid containing the respective gene fused to an eGFP tag, controlled by the Pgpd promoter (a strong, constitutive promoter), was introduced into the corresponding strains via transformation.Ascospores were isolated from the transformants that reached fertility, allowing the acquisition of homokaryotic strains carrying the complementation construct.

Growth tests and genotoxic stress assays
Growth tests were performed on BMM media and the growth front was documented every 24 h.All tests were performed with three biological replicates.Statistical evaluation was conducted using a Student's t-test.Phenotypic characterization involved observing and documenting the growth and fertility of the strains on BMM plates using a Stemi 2000-C stereomicroscope (Zeiss).To assess the resistance against MMS, all strains were inoculated on BMM plates supplemented with 0.007% (v/v) MMS, and the growth was observed and documented over a period of 4 days.Similarly, resistance against HU was evaluated on BMM plates containing 8 mM HU using the same approach.

Western blot analysis
To evaluate differences in the global levels of H3K56ac between the Δrtt109 strains and the wild-type, a western blot analysis was conducted using antibodies specific to this particular modification.The wild-type strain SN1693, the Δrtt109 strain S60, and the corresponding complementation strain S60K1 were grown in liquid BMM medium (Esser 1982) at 27 °C for 4 days.The mycelium was harvested by filtration, washed with an isotonic buffer established for protoplasts (Walz and Kück 1995) and subsequently frozen in liquid nitrogen before being ground to a fine powder.Protein extraction was performed by mixing the powder with extraction buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.05% NP-40, 0.3% (v/v) Protease Inhibitor Cocktail Set IV (Calbiochem)).After 20 min centrifugation, the protein concentrations were determined using Bradford assays (Bradford 1976).Equal amounts of proteins were separated by SDS gel electrophoresis, and transferred onto a PVDF membrane through western blotting.H3K56ac-specific antibodies (Active Motif #39082) and H3-specific antibodies (Cell Signaling #9715) were used to detect the respective bands.

Deletion of rtt109 in S. macrospora leads to sterility and impairs vegetative growth
For the functional characterization of the histone acetyltransferase RTT109 in S. macrospora, a ku70 deletion strain was transformed using a deletion cassette designed for the rtt109 sequence.The deletion cassette consisted of a hygromycin phosphotransferase gene flanked by sequences homologous to the rtt109 gene, enabling the replacement of the target gene with a resistance marker.Primary transformants that exhibited resistance to hygromycin were selected and subsequently crossed in order to obtain homokaryotic deletion mutants without the Δku70 background.The deletion of rtt109 in the resulting strains was verified by PCR with primers designed to amplify a part of the gene of interest.The correct integration of the deletion cassette was verified by PCR (Supplementary Fig. 1).Throughout the generation of the deletion strains, primary transformants did not exhibit any noticeable defects in sexual development and demonstrated the ability to achieve fertility.Since primary transformants are heterokaryotic in most cases, effects of gene deletions are often not directly obvious and sometimes appear only in homokaryotic strains.To obtain homokaryons, the primary transformants were crossed with the spore color mutant fus and the resulting homokaryotic ascospores were isolated.Homokaryotic Δrtt109 strains were found to be sterile (Supplementary Table 4).Microscopic examination confirmed a block in the sexual development of Δrtt109 at the stage of young protoperithecia (Fig. 1).While the ascogonia and early protoperithecia were formed normally and at the expected time, no further structures of sexual development were observed.Even after a week, there was no developmental progression beyond that stage.The developmental effects of rtt109 deletions in S. macrospora appeared to be even more severe than in other ascomycete model organisms such as F. graminearum, where a reduction in ascospore formation and aberrant perithecia have been previously documented (Kong et al. 2018).rtt109 fused with a C-terminal eGFP tag was reintroduced into the validated deletion strains through ectopic integration to conduct complementation analysis and perform localization studies using fluorescence microscopy.Complementation strains regained fertility and were able to grow wild-type-like sexual structures (Fig. 1).
Deletion of rtt109 also inhibited the vegetative growth rate of the respective mutants.Since RTT109 is suspected to be an interaction partner of ASF1, we documented the growth of rtt109 deletion mutants, the respective complementation strain, and asf1 deletion mutants to compare them to each other and to the wildtype (Fig. 2).Deletion of rtt109 caused a visible reduction in vegetative growth rate, which was very similar to that of asf1 deletion mutants, although the density of Δrtt109 mycelium appeared higher and more wild-type-like.The growth rate of rtt109 deletion strains was fully restored to wild-type levels by reintroduction of the rtt109 gene (Fig. 2).We quantified this observation by measuring the progress of the growth front in five biological replicates and detected a reduction of vegetative growth speed of around 40% in Δrtt109 and Δasf1 strains (Fig. 3).The impairment of vegetative growth in S. macrospora Δrtt109 is consistent with results in F. graminearum, where corresponding deletion mutants were also reported to grow 40% slower than the wild-type (Kong et al. 2018).This effect was completely reversed by complementation of the rtt109 mutant and the respective strains exhibited a growth speed comparable to the wild-type (Fig. 3).In summary, the findings indicate that rtt109 plays a crucial role in the sexual development of S. macrospora and is also significant for vegetative growth.The phenotype of rtt109 deletion mutants appears highly similar to that of asf1 deletion mutants, suggesting that indeed both function in the same pathway.
To further assess whether asf1 and rtt109 act in a similar manner during development, or whether there is a combinatorial effect in the absence of both genes, we attempted to cross Δasf1 and Δrtt109 strains to obtain double mutants.However, no fruiting bodies were formed in crosses of the two strains (Supplementary Fig. 2).The inability of the mutants to cross Chromatin-associated proteins in fungi | 3 with each other may be another indication that ASF1 and RTT109 act in a similar developmental pathway.

S. macrospora Δrtt109 and Δasf1 react similarly to MMS and HU
In previous work, we discovered a sensitivity of S. macrospora Δasf1 strains to the DNA damaging agent MMS as well as a reduction in global H3K56ac levels in Δasf1 (Breuer et al. 2024).The histone acetyltransferase RTT109 interacts with the chromatin modifier ASF1 in Candida albicans (Lercher et al. 2018), Schizosaccharomyces pombe (Han, Zhou, Li et al. 2007), and S. cerevisiae (Tsubota et al. 2007) and is involved in various mechanisms that ensure DNA stability during replication.Additionally, RTT109 is known as the primary enzyme responsible for H3K56 acetylation in S. cerevisiae (Han, Zhou, Horazdovsky et al. 2007).In the case of A. fumigatus, deletion of rtt109 led to heightened sensitivity to DNA damaging agents such as MMS and HU (Zhang et al. 2021).MMS induces DNA methyl adducts, which can cause double-strand breaks during replication (Ensminger et al. 2014), while HU inhibits the ribonucleotide reductase and leads to replication arrest by affecting dNTP supply (Koç et al. 2004).In our study, we exposed S. macrospora Δrtt109, the corresponding complementation strain, an asf1 deletion mutant, and the wild-type to MMS and HU.We found that the deletion of rtt109 resulted in severe sensitivity to MMS, comparable to the sensitivity exhibited by the asf1 mutant (Fig. 4).While the wild-type and complementation strains were able to grow relatively normally on BMM media containing MMS, the mutants failed to grow at all.Strikingly, we did not observe increased sensitivity against HU for the mutant strains.While aerial hyphae production appeared somewhat reduced, all strains were able to grow under HU stress (Fig. 5).These observations contrast with the effects of rtt109 deletions in S. cerevisae (Han, Zhou, Horazdovsky et al. 2007) and A. fumigatus (Zhang et al. 2021), where rtt109 mutants are sensitive to MMS as well as HU, suggesting the presence of additional factors in S. macrospora that respond to such specific stress conditions.

S. macrospora RTT109 is localized in the nucleus
Given that RTT109 is described as a histone acetyltransferase, its targets are expected to be found within the nuclear compartment of the cell.Consequently, it is necessary for RTT109 to localize within the nucleus.Our complementation experiments with rtt109 fused to an eGFP tag restored the phenotype of the deletion mutant, so a correct localization of the fusion protein was expected.To analyze the localization of RTT109, we employed fluorescence microscopy on the complementation strains.These strains were cultivated alongside a marker strain that expressed histone H3 fused to an mRFP tag.Since histones are primarily localized in the nucleus, this setup enabled us to perform colocalization analysis in the resulting heterokaryons (Schmidt et al. 2020).Through the detection of both green and red fluorescence in the same cellular compartment, we confirmed the colocalization of RTT109 and histone H3 (Fig. 6).Based on these findings, we can conclude that RTT109 in S. macrospora is indeed localized within the nucleus.

The rtt109 deletion leads to a loss of H3K56ac in S. macrospora
RTT109 is widely recognized as the primary, and possibly exclusive, histone acetyltransferase responsible for the acetylation of 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.
H3K56 in fungal organisms (Driscoll et al. 2007;Tsubota et al. 2007;Han, Zhou, Horazdovsky et al. 2007).This specific histone modification has been demonstrated to play a crucial role in the cellular response to DNA damage induced by MMS (Wurtele et al. 2012).Therefore, we analyzed the levels of H3K56ac in the S. macrospora Δrtt109 mutant.To assess changes in the levels of global H3K56ac, we conducted western blot analysis in the S. macrospora Δrtt109 deletion mutant and compared the results to the wild-type and the complemented mutant.Our findings revealed a loss of this histone modification in the deletion mutant (Fig. 7, Supplementary Fig. 3).The complementation strain exhibited a complete restoration of H3K56ac production, indicating the successful reinstatement of normal acetylation.

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.

Deletion of chk2, a functional equivalent of rad53, has no effect on sexual development
The second potential partner for ASF1 during DNA damage protection we analyzed was the putative checkpoint kinase CHK2, which is orthologous to the checkpoint kinases PRD-4 from N. crassa and the human CHK2.It is the closest S. macrospora homolog to the S. cerevisiae checkpoint kinase Rad53 (Supplementary Fig. 4).In contrast to the analyzed filamentous fungi and animals, there is a second homolog in S. cerevisiae, Dun1, which is more similar to PRD-4 and CHK2 proteins from animals both in sequence and domain structure than Rad53 (Supplementary Fig. 4b).However, PRD-4 from N. crassa and the human CHK2 were both shown to be able to complement an S. cerevisiae Δrad53 mutant and are therefore functionally equivalent to Rad53 (Matsuoka et al. 1998;Pregueiro et al. 2006).Rad53 plays a critical role in maintaining genomic stability and regulating cell cycle arrest during DNA damage repair, as well as histone recycling in S. cerevisiae (Gunjan and Verreault 2003).
In yeast, the interaction between Asf1 and Rad53 is well documented, with Asf1 binding hypophosphorylated, inactive Rad53 and thus ensuring recovery from DNA damage repair (Tsabar et al. 2016).To investigate the role of CHK2 in S. macrospora, we deleted the S. macrospora chk2 using homologous recombination.Hygromycin resistant primary transformants were crossed to obtain homokaryotic strains, and the deletion of chk2 was confirmed by PCR (Supplementary Fig. 5).Both primary transformants and homokaryotic deletion mutants showed normal growth and fruiting body development without any noticeable differences compared to the wild-type.Vegetative growth appeared wild-type-like (Fig. 8) and Δchk2 strains achieved fertility within the expected timeframe.To further assess potential developmental defects, we examined the morphology of perithecia and asci in the mutant compared to the wild-type and found no detectable differences (Fig. 9).Thus, a deletion of chk2 has no discernible effect on the development of S. macrospora.

S. macrospora Δchk2 shows the opposite reaction to MMS and HU as Δrtt109 and Δasf1
Given the known involvement of RAD53 and its functional equivalent PRD-4 in DNA damage protection (Pellicioli et al. 1999;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.Wakabayashi et al. 2008), it was reasonable to expect a similar role for S. macrospora CHK2.Therefore, we conducted sensitivity tests with the genotoxic substances MMS and HU on the S. macrospora chk2 deletion mutant.Surprisingly, the presence or absence of chk2 did not have a noticeable impact on MMS resistance in S. macrospora (Fig. 10), in contrast to findings with the N. crassa ortholog PRD-4 (Pregueiro et al. 2006).This suggests that the relationship between ASF1 and CHK2 may not be significant in the context of MMS-induced stress in our model system.To evaluate the possibility that a CHK2 paralog might exist in S. macrospora that could substitute for CHK2 in the Δchk2 mutant and thus explain the lack of developmental phenotypes and the MMS resistance of the mutant, we performed BLASTP analysis (Altschul et al. 1997) with CHK2.While several proteins showed partial similarity to CHK2, this similarity was restricted to the serine/ threonine protein kinase domain, and none of the proteins contained the additional forkhead-associated domain that is present in CHK2 orthologs from S. macrospora and other fungi (Supplementary Figs.4A, 6, and 7).Thus, it appears unlikely that these putative kinases can substitute for CHK2 in the Δchk2 mutant.
In contrast to growth on MMS, under HU stress, the deletion of chk2 had a clear effect, unlike the deletion of asf1 (Fig. 11).Reintroduction of chk2 into S. macrospora Δchk2 strains complemented the HU sensitivity phenotype (Supplementary Table 5).While all strains initially exhibited slow growth under HU stress without significant differences within the first 48 h, Δchk2 mutants ceased growing after 48 h.In contrast, the wild-type and asf1 deletion strain proved to be resistant, consistent with previous observations comparing them to Δrtt109 strains.These results suggest that CHK2 plays a critical role in the DNA damage response pathway specific to coping with HU-induced stress, independent of ASF1 or RTT109.

RTT109 might be necessary for numerous functions of ASF1 during DNA damage protection and sexual development
In this study, we investigated the role of two potential partners of the histone chaperone ASF1 during DNA damage protection and sexual development in the ascomycete S. macrospora.Our findings suggest that the histone acetyltransferase RTT109 has significant functions under normal and DNA damage stress conditions.Deletion of rtt109 resulted in strains that exhibited similarities to asf1 deletions in terms of vegetative growth, sexual development, histone acetylation, and DNA damage response.These observations suggest a close relationship between ASF1 and RTT109 in these processes, as supported by their documented interactions in other fungal models like C. albicans, S. pombe, and S. cerevisae (Tsubota et al. 2007;Han, Zhou, Li et al. 2007;Lercher et al. 2018).S. macrospora rtt109/asf1 double mutants may be useful to determine if the two proteins act in the same pathway.A phenotype similar to single mutants with respect to growth and development should be observed in double mutants if RTT109 and ASF1 act in a cooperative manner during these processes.However, the single mutants proved to be unable to form sexual structures in genetic crosses.This could be the result of an effect on sexual development shared by both deletion strains, or the result of stress levels too high to allow proper crossing.The similarities between the respective deletion strains described raise the hypothesis that the severe phenotype observed in S. macrospora Δasf1 may be the consequence of the lack of interaction with RTT109.Studies in budding yeast have shown that Rtt109 relies on Asf1 to obtain histone H3 for acetylating K56 and the absence of Asf1 may nullify the functions of Rtt109 (Zhang et al. 2018).In a previous work, we demonstrated that sexual development in S. macrospora depends on the ability of ASF1 to bind histones and hypothesized that it acts as scaffold for H3-H4 interactions with other chromatin modifiers (Breuer et al. 2024).The absence of such a scaffold might impair the ability of RTT109 to perform its functions in histone acetylation, which could cause the severe developmental defects in asf1 deletion mutants.Thus, a reduction or improper positioning of H3K56ac may be the underlying issue in S. macrospora Δasf1 and proper acetylation of H3 may be crucial for sexual development.While overexpression experiments with rtt109 in S. macrospora Δasf1 might provide information about the general level of H3K56ac necessary for development, positioning might still be problematic, and since ASF1 is thought to be the scaffold necessary for RTT109 to function at its full extent (Cote et al. 2019), higher RTT109 levels might not even correlate with higher H3K56ac levels in asf1 deletion mutants.However, given the wide range of putative and proven functions of ASF1 (Mousson et al. 2007), it may be challenging to pinpoint its role during S. macrospora development to a single interaction partner and its effect on H3K56ac.Interestingly, the DNA damage response functions of ASF1 do not seem to depend on its interaction with histones in the same way as sexual development, although ASF1 variants unable to bind histones showed a similar reduction of H3K56ac as full deletion mutants (Breuer et al. 2024).However, our results on the sensitivity of rtt109 deletion mutants to MMS show a dependence on RTT109 for DNA damage protection and therefore probably for the establishment of H3K56ac.More sensitive assays such as ChIP-seq are needed to fully quantify the potential differences in H3K56ac levels between S. macrospora Δasf1 and Δrtt109, and our results indicate that deletion of the HAT causes an even greater decrease, or even complete loss in H3K56 acetylation levels than the loss of a putative scaffold.In S. cerevisiae, Rtt109 is known to interact not only with Asf1 but also with another histone chaperone, Vps75, which also binds H3 and H4 (Tsubota et al. 2007).Although a fully functional Asf1-Vps75-Rtt109 complex is known to be necessary for establishing correct acetylation patterns in budding yeast (Adkins et al. 2007), the mere presence of ASF1, even without histone binding ability, might be sufficient to enable lower-level activity of RTT109, providing some form of DNA damage protection.Therefore, the severe developmental defects observed in S. macrospora Δasf1, persistent when expressing ASF1 variants unable to bind histones, may have roots not only in the misregulation of developmental processes, but also in the accumulation of underlying DNA damage events, compromising the respective strains as a whole.Such DNA damage accumulation might be too insignificant to turn up during a sensitivity assay, but could potentially lead to disturbances during tightly regulated processes, such as sexual development and fruiting body formation.While this hypothesis requires further investigation, such as interaction studies between RTT109, ASF1 and VPS75, the role of RTT109 during sexual development and the DNA damage response in S. macrospora appears to be quite fundamental.As the primary facilitator of H3K56ac in fungi (Han, Zhou, Horazdovsky et al. 2007), RTT109 is clearly essential for survival under MMS conditions, which can induce double-strand breaks during replication (Ensminger et al. 2014).Furthermore, RTT109 is crucial during the formation of complex sexual structures, either by providing the necessary genomic stability for such processes or by ensuring the correct transcription of important genes through the establishment of proper acetylation patterns.Surprisingly, the deletion of rtt109 did not increase the sensitivity of S. macrospora to HU, despite its well-known DNA damaging properties that cause replication fork arrest by disrupting dNTP availability (Koç et al. 2004).In contrast, other fungal model organisms like A. fumigatus and S. cerevisiae have been shown to exhibit heightened sensitivity to HU when rtt109 is deleted (Zhang et al. 2021;Cañas et al. 2022).

CHK2 is not essential for sexual development, but provides resistance to genotoxic stress independent of ASF1
The second putative partner of ASF1 during DNA damage protection we investigated was CHK2.CHK2 is the ortholog of N. crassa 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.
Chromatin-associated proteins in fungi | 9 PRD-4, which can complement a Δrad53 mutant with respect to its function in DNA damage response in S. cerevisiae (Pregueiro et al. 2006).Deletion of chk2 did not result in any noticeable defects in vegetative growth or developmental processes, suggesting its negligible role in the formation of fruiting bodies.Furthermore, the mutants lacking chk2 did not exhibit increased sensitivity to MMS-induced DNA damage stress, unlike the highly sensitive asf1 and rtt109 mutants.This indicates that CHK2 operates in a  distinct damage response pathway from ASF1 and RTT109 under these specific conditions.However, we discovered a role for CHK2 in response to a different type of genotoxic stress, as the mutants showed sensitivity to the replication fork stressor hydroxyurea, while the asf1 and rtt109 mutants did not display such sensitivity.The budding yeast equivalent, Rad53, is known to be important for restarting inhibited replication forks (Szyjka et al. 2008), so it is reasonable to assume that the replication fork stalling caused by HU cannot be efficiently rescued in the absence of Rad53, or in this case CHK2 in S. macrospora.Since Δasf1 strains are not inhibited under HU stress, this suggests that CHK2 is involved in a genomic protection system that functions independently of ASF1.Notably, this observation appears to be specific for S. macrospora, as an S. cerevisiae Δasf1 mutant is sensitive to HU (Lin et al. 2010).The precise functions of CHK2 and its relationship with ASF1 in its broader chromatin modification network are more challenging to elucidate, given the specific roles fulfilled by homologous checkpoint kinases like PRD-4 or functional equivalents like RAD53 in their respective native organisms.However, it can be inferred that any connection between ASF1 and CHK2 in DNA damage protection and sexual development is of minor importance.Additionally, the diverse functions of ASF1 in S. macrospora do not seem to include an active role in the response to HU-induced stress.
In conclusion, our study revealed an essential role of the histone acetyltransferase RTT109 during sexual development, vegetative growth, histone modification, and genotoxic stress response in the ascomycete S. macrospora.The phenotypic aberrations in rtt109 deletion strains closely resemble those previously observed in asf1 deletion mutants, indicating a strong correlation between the functions of ASF1 and RTT109.CHK2, another potential component of the ASF1-mediated chromatin modifier network, appears to be unrelated to these processes but is instead involved in a distinct DNA damage response system that operates independently of ASF1.

Fig. 5 .
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 .
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. 9 .
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 .
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.