Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response.

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.

Fun30 and Rtt109 Mediate Epigenetic Regulation of the DNA Damage Response Pathway in C. albicans.

Fun30, an ATP-dependent chromatin remodeler from S. cerevisiae, is known to mediate both regulation of gene expression as well as DNA damage response/repair. The Fun30 from C. albicans has not yet been elucidated. We show that C. albicans Fun30 is functionally homologous to both S. cerevisiae Fun30 and human SMARCAD1. Further, C. albicans Fun30 can mediate double-strand break end resection as well as regulate gene expression. This protein regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathways. The regulation mediated by C. albicans Fun30 is dependent on its ATPase activity. The expression of FUN30, in turn, is regulated by histone H3K56 acetylation catalyzed by Rtt109 and encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild-type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1, and RAD5 expression; ChIP experiments showed that Fun30 but not H3K56ac regulates the expression of these genes in response to oxidative stress. In contrast, upon treatment with MMS, the expression of RAD9 is upregulated, which is modulated by both Fun30 and H3K56 acetylation. Thus, Fun30 and H3K56 acetylation mediate the response to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

Crystal structure of histone chaperone Vps75 from Candida albicans.

Vps75 is a histone chaperone that interacts with the fungal-specific histone acetyltransferase Rtt109 and stimulates its acetylation activity on histone H3. Here we report the crystal structure of Vps75 of Candida albicans, one of the most common fungal pathogens. CaVps75 exists as a headphone-like dimer that forms a large negatively charged region on its concave side, showing the potential to bind positively charged regions of histones. The distal ends of the concave side of the CaVps75 dimer are positively charged and each has one more α helix than yeast Vps75. CaVps75 exhibits ionic strength- and concentration-dependent higher oligomerization in solution. In the crystal, two dimers are bound through electrostatic interactions between charged regions on the concave side of their earmuff domains, and this inter-dimer interaction differs from the currently known inter-dimer interactions of Vps75s. Our results will help to understand the role of Vps75 in C. albicans.

The Fungi-specific histone Acetyltransferase Rtt109 mediates morphogenesis, Aflatoxin synthesis and pathogenicity in Aspergillus flavus by acetylating H3K9.

Aspergillus flavus is a common saprophytic filamentous fungus that produces the highly toxic natural compound aflatoxin during its growth process. Synthesis of the aflatoxins, which can contaminate food crops causing huge losses to the agricultural economy, is often regulated by epigenetic modification, such as the histone acetyltransferase. In this study, we used Aspergillus flavus as an experimental model to construct the acetyltransferase gene rtt109 knockout strain (△rtt109) and its complementary strain (△rtt109·com) by homologous recombination. The growth of △rtt109 was significantly suppressed compared to the wild type (WT) strain and the △rtt109·com strain. The sclerotium of △rtt109 grew smaller, and the amount of sclerotia generated by △rtt109 was significantly reduced. The number of conidiums of △rtt109 was significantly reduced, especially on the yeast extract sucrose (YES) solid medium. The amount of aflatoxins synthesized by △rtt109 in the PDB liquid medium was significantly decreased We also found that the △rtt109 strain was extremely sensitive to DNA damage stress. Through the maize seed infection experiment, we found that the growth of △rtt109 on the surface of affected corn was largely reduced, and the amount of aerial mycelium decreased significantly, which was consistent with the results on the artificial medium. We further found that H3K9 was the acetylated target of Rtt109 in A. flavus. In conclusion, Rtt109 participated in the growth, conidium formation, sclerotia generation, aflatoxin synthesis, environmental stress response, regulation of infection of A. flavus. The results from this study of rtt109 showed data for acetylation in the regulation of life processes and provided a new thought regarding the prevention and control of A. flavus hazards.

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats.

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

Transcription levels of a noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast.

Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes.

The fungal-specific histone acetyltransferase Rtt109 regulates development, DNA damage response, and virulence in Aspergillus fumigatus.

In eukaryotes, histone acetylation catalyzed by histone acetyltransferase (HAT) has been demonstrated to be critical for various physiological processes. However, the biological functions of HAT and the underlying mechanism by which HAT-regulated processes are involved in fungal development and virulence in the human opportunistic pathogen Aspergillus fumigatus remain largely unexplored. Here, we functionally characterized the roles of Rtt109 in A. fumigatus, an ortholog of Saccharomyces cerevisiae histone acetyltransferase Rtt109. In vivo and in vitro HAT assays revealed that AfRtt109 functions as a canonical histone acetyltransferase, acetylating lysines 9 and 56 of histone H3. Deletion of Afrtt109 leads to severe defects in vegetative growth, conidiation, and causes reduced virulence in the Galleria mellonella model, as well as hypersensitivity to genotoxic agents. Moreover, site-directed mutagenesis revealed that the conserved arginine residues R265 and R306 of Rtt109 are required for the H3K9 and H3K56 acetylation and virulence of A. fumigatus. Unexpectedly, R265E and R306E mutants did not exhibit any detectable phenotypic defects, implying that A. fumigatus Rtt109 regulates fungal development via histone acetylation-independent mechanisms. Together, our results revealed the critical role of fungal-specific HAT Rtt109 in regulating fungal development and virulence, and suggested that it may serve as a unique target for antifungal therapies.

A role for Rtt109 in buffering gene-dosage imbalance during DNA replication.

Chromatin can function as an integrator of DNA-related processes, allowing communication, for example, between DNA replication and gene transcription. Such communication is needed to overcome the gene-dosage imbalance introduced during DNA replication, when certain genes are replicated prior to others. Increased transcription of early replicating genes could alter regulatory balances. This does not occur, suggesting a mechanism that suppresses expression from newly replicated DNA. Critical to this buffering is Rtt109, which acetylates the internal K56 residue of newly synthesized histone H3 prior to incorporation onto DNA. H3K56ac distinguishes replicated from non-replicated DNA, communicating this information to the transcription machinery to ensure expression homeostasis during S phase.

Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae.

Protein kinase C coordinates histone H3 phosphorylation and acetylation.

The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly.

Role of the rttA gene in morphogenesis, stress response, and virulence in the human pathogenic fungus Penicillium marneffei.

Penicillium marneffei is a human pathogenic fungus and the only thermally dimorphic species of the genus. At 25°C, P. marneffei grows as a mycelium that produces conidia in chains. However, when incubated at 37°C or following infection of host tissue, the fungus develops as a fission yeast. Previously, a mutant (strain I133) defective in morphogenesis was generated via Agrobacterium-mediated transformation. Specifically, the rtt109 gene (subsequently designated rttA) in this mutant was interrupted by T-DNA insertion. We characterized strain I133 and the possible roles of the mutated rttA gene in altered P. marneffei phenotypes. At 25°C, the rttA mutant produces fewer conidia than the wild type and a complemented mutant strain, as well as slower rates of conidial germination; however, strain I133 continued to grow as a yeast in 37°C-incubated cultures. Furthermore, whereas the wild type exhibited increased expression of rttA at 37°C in response to the DNA-damaging agent methyl methane sulfonate, strain I133 was hypersensitive to this and other genotoxic agents. Under similar conditions, the rttA mutant exhibited decreased expression of genes associated with carbohydrate metabolism and oxidative stress. Importantly, when compared with the wild-type and the complemented strain, I133 was significantly less virulent in a Galleria infection model when the larvae were incubated at 37°C. Moreover, the mutant exhibited inappropriate phase transition in vivo. In conclusion, the rttA gene plays important roles in morphogenesis, carbohydrate metabolism, stress response, and pathogenesis in P. marneffei, suggesting that this gene may be a potential target for the development of antifungal compounds.

Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.