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.

Stimulation of adaptive gene amplification by origin firing under replication fork constraint

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32, factors known to alter the frequency of template switching during break induced replication (BIR). CUP1 amplification is also critically dependent on late firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after firing, leaving an epigenetic scar of H3K56 acetylation that promotes template switching during later break induced replication events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

The Amazing Acrobat: Yeast’s Histone H3K56 Juggles Several Important Roles While Maintaining Perfect Balance

Acetylation on lysine 56 of histone H3 of the yeast Saccharomyces cerevisiae has been implicated in many cellular processes that affect genome stability. Despite being the object of much research, the complete scope of the roles played by K56 acetylation is not fully understood even today. The acetylation is put in place at the S-phase of the cell cycle, in order to flag newly synthesized histones that are incorporated during DNA replication. The signal is removed by two redundant deacetylases, Hst3 and Hst4, at the entry to G2/M phase. Its crucial location, at the entry and exit points of the DNA into and out of the nucleosome, makes this a central modification, and dictates that if acetylation and deacetylation are not well concerted and executed in a timely fashion, severe genomic instability arises. In this review, we explore the wealth of information available on the many roles played by H3K56 acetylation and the deacetylases Hst3 and Hst4 in DNA replication and repair.

Histone Acetyltransferase p300 Inhibitor Improves Coronary Flow Reserve in SIRT3 (Sirtuin 3) Knockout Mice

Background Coronary microvascular dysfunction is common in patients of myocardial infarction with non‐obstructive coronary artery disease. Coronary flow reserve (CFR) reflects coronary microvascular function and is a powerful independent index of coronary microvascular dysfunction and heart failure. Our previous studies showed that knockout of SIRT3 (Sirtuin 3) decreased CFR and caused a diastolic dysfunction. Few studies focus on the treatment of impaired CFR and heart failure. In the present study, we explored the role of C646, a histone acetyltransferase p300 inhibitor, in regulating CFR and cardiac remodeling in SIRT3 knockout (SIRT3KO) mice. Methods and Results After treating with C646 for 14 days, CFR, pulse‐wave velocity, and cardiac function were measured in SIRT3KO mice. SIRT3KO mice treated with C646 showed a significant improvement of CFR, pulse‐wave velocity, ejection fraction, and fractional shortening. Treatment with C646 reversed pre‐existing cardiac fibrosis, hypertrophy, and capillary rarefaction in SIRT3KO mice. Mechanistically, knockout of Sirtuin 3 resulted in significant increases in p300 expression and H3K56 acetylation. Treatment with C646 significantly reduced levels of p300 and H3K56 acetylation in SIRT3KO mice. Furthermore, treatment with C646 increased endothelial nitric oxide synthase expression and reduced arginase II expression and activity. The expression of NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) and VCAM‐1 (vascular cell adhesion molecule 1) was also significantly suppressed by C646 treatment in SIRT3KO mice. Conclusions C646 treatment attenuated p300 and H3K56 acetylation and improved arterial stiffness and CFR via improvement of endothelial cell (EC) dysfunction and suppression of NF‐κB.

Potential Functions of Histone H3.3K56 Acetylation in Mammals

H3K56 acetylation (H3K56Ac) was first identified in yeast and has recently been reported to play important roles in maintaining genomic stability, chromatin assembly, DNA replication, cell cycle progression and DNA repair. Although H3.1K56Ac has been relatively well studied, the function of H3.3K56Ac remains mostly unknown in mammals. In this study, we used H3.3K56Q and H3.3K56R mutants to study the possible function of H3.3K56 acetylation. The K-to-Q substitution mimics a constitutively acetylated lysine, while the K-to-R replacement mimics a constitutively unmodified lysine. We report that cell lines harboring mutation of H3.3K56R exhibit dramatic morphology changes and cell death. Using Tet-Off inducible system, we show an increased population of polyploid/aneuploid cells and a decreased cell viability in H3.3K56R mutant cells. In consistence with these results, H3.3K56R mutant compromised H3.3 incorporation into several pericentric and centric heterochromatin regions we tested. Moreover, mass spectrometry analysis coupled with label free quantification reveal that biological processes regulated by the H3.3-associating proteins, whose interaction with H3.3 is markedly increased by H3.3K56Q mutation but decreased by H3.3K56Q mutation, include sister chromatid cohesion, mitotic nuclear division, and mitotic nuclear envelope disassembly. These results suggest that H3.3K56 acetylation is crucial for chromosome segregation and cell division in mammals.

Role of the Histone Acetyltransferase Rtt109 in Development and Pathogenicity of the Rice Blast Fungus

Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 plays important roles in maintaining genome integrity and surviving DNA damage. Here, we investigated the implications of Rtt109-mediated response to DNA damage on development and pathogenesis of the rice blast fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae). The ortholog of Rtt109 in M. oryzae (MoRtt109) was found via sequence homology and its functionality was confirmed by phenotypic complementation of the Saccharomyces cerevisiae Rtt109 deletion strain. Targeted deletion of MoRtt109 resulted in a significant reduction in acetylation of H3K56 and rendered the fungus defective in hyphal growth and asexual reproduction. Furthermore, the deletion mutant displayed hypersensitivity to genotoxic agents, confirming the conserved importance of Rtt109 in genome integrity maintenance and genotoxic stress tolerance. Elevated expression of DNA repair genes and the results of the comet assay were consistent with constitutive endogenous DNA damage. Although the conidia produced from the mutant were not impaired in germination and appressorium morphogenesis, the mutant was significantly less pathogenic on rice leaves. Transcriptomic analysis provided insight into the factors underlying phenotypic defects that are associated with deficiency of H3K56 acetylation. Overall, our results indicate that MoRtt109 is a conserved histone acetyltransferase that affects proliferation and asexual fecundity of M. oryzae through maintenance of genome integrity and response to DNA damage.