Fungal enzyme RTT109 proves essential for inheriting silenced chromatin

Every time a cell divides, it faces a formidable copying problem that goes far beyond DNA. The genome is wrapped around histone proteins and decorated with chemical marks that tell the cell which genes to keep silent, and each round of replication dilutes those marks as new, unmodified histones are deposited onto freshly copied DNA. How repressed chromatin states survive this disruption is one of the central puzzles of epigenetics. A new study in the model fungus Neurospora crassa, published in PLOS Genetics by Rochelle Yap, Felicia Ebot-Ojong, Abigail Ameri-Solanky and Zachary Lewis of the University of Georgia, identifies an unexpected guardian of this memory: the replication-associated histone acetyltransferase RTT109.

Polycomb Repressive Complex 2, or PRC2, is the enzyme responsible for writing one of biology’s most important repressive marks, the trimethylation of histone H3 at lysine 27, known as H3K27me3. In animals and plants, this mark silences developmental genes and helps cells remember what they are supposed to become. Once PRC2 establishes H3K27me3, the mark can spread along chromosomes through a read-write mechanism in which the complex recognizes the modification it has just deposited and methylates neighboring nucleosomes. Remarkably, in mammals these repressed domains can persist through many rounds of cell division even when the DNA sequences that originally recruited PRC2 are no longer needed, implying that the mark itself carries heritable information.

Fungi present an intriguing simplification of this picture. Polycomb Repressive Complex 1, which in plants and animals helps recruit and reinforce PRC2, was apparently lost early in fungal evolution, yet many filamentous fungi still assemble large H3K27me3 domains that stably repress condition-specific genes. In Neurospora crassa, these domains cover roughly seven percent of the genome and cluster together in three-dimensional nuclear space. The fungus therefore offers a clean experimental system for asking whether H3K27me3 can be inherited across mitosis without PRC1, and which factors make that inheritance possible.

The Georgia team began with a genetic screen, searching RNA-sequencing datasets for knockout strains that had lost repression of H3K27me3-marked genes. RTT109 stood out. This enzyme was originally discovered in budding yeast as a regulator of Ty1 transposition and is best known for acetylating histone H3 at lysine 56 on newly synthesized histones during replication-coupled nucleosome assembly, the process by which old histones are recycled and new ones deposited behind the replication fork. When the researchers deleted the rtt109 gene, a reporter gene embedded in a facultative heterochromatin domain switched on, mimicking the effect of destroying PRC2 itself.

Transcriptomic analysis confirmed the breadth of the defect. Of 573 genes normally marked with H3K27me3, 136 were significantly upregulated in the rtt109 deletion strain, and the misexpressed genes were strongly enriched for functions normally activated during fruiting body development, including fungal cell wall synthesis and chitin metabolism. Importantly, deleting other histone acetyltransferases did not reproduce this pattern, showing that the effect is specific to RTT109 rather than a generic consequence of altered histone acetylation. Across the whole genome, 705 of 722 differentially expressed genes were upregulated, consistent with a loss of repression rather than a broad transcriptional collapse.

Chromatin immunoprecipitation sequencing revealed that the transcriptional defects were accompanied by structural erosion of the repressed domains themselves. In the deletion strain, 102 of 281 H3K27me3 domains lost at least half of their signal, and the median domain size shrank from 6.1 kilobases in wild type to 4.5 kilobases. The losses were regional rather than uniform: domains near telomeres, which act as strong recruitment sites for PRC2, retained or even gained the mark, while losses increased with distance from chromosome ends. Introducing a tagged wild-type copy of rtt109 restored both the methylation pattern and the DNA-damage repair defect characteristic of the mutant, confirming that RTT109 was directly responsible.

The team then dissected the mechanism with a series of elegant controls. RTT109 is known to work with histone chaperones, and affinity purification coupled to mass spectrometry identified NAF-2, the Neurospora homolog of yeast Vps75, as a high-confidence interactor, with AlphaFold 3 predicting a detailed contact interface between the two proteins. Yet deleting naf-2 did not disrupt H3K27me3 or gene repression, and neither did reducing levels of the essential chaperone ASF-1. More strikingly, histone H3 mutants that either abolish or mimic acetylation at lysine 56, RTT109’s canonical substrate, left H3K27me3 patterns essentially intact. In contrast, engineered catalytically dead versions of RTT109 failed to rescue the methylation defects and even worsened them, suggesting a dominant-negative effect. The conclusion is that RTT109’s enzymatic activity is required for Polycomb-repressed chromatin, but its famous product, H3K56 acetylation, is not the relevant output.

To test whether RTT109 safeguards epigenetic memory over time, the researchers passaged a freshly backcrossed deletion strain through successive rounds of asexual reproduction, each passage representing roughly 25 to 30 mitotic divisions. H3K27me3 declined progressively with each passage, indicating that the mark decays without RTT109 rather than being lost instantly. A second repressive mark, H3K36me3 deposited by the methyltransferase ASH1 within the same facultative heterochromatin domains, was lost much more rapidly, appearing already depleted in the earliest isolate. Because ASH1-dependent H3K36 methylation is known to support gene silencing and proper H3K27me3 patterning in Neurospora, the authors propose that its early loss may trigger the slower decay of H3K27me3 downstream.

The most decisive experiment used a synthetic system to separate establishment from maintenance. The team tethered a PRC2 subunit, SUZ12, to an array of tet operator sequences inserted at a defined genomic locus, allowing them to direct de novo H3K27me3 deposition at will. Adding tetracycline released the tether, and the researchers could then ask whether the native PRC2 complex could maintain the mark without sequence-specific recruitment. In wild-type cells, H3K27me3 persisted at the array for at least 24 hours, an interval estimated to span 10 to 12 rounds of nuclear division, demonstrating true epigenetic inheritance in an organism that lacks PRC1. In cells lacking RTT109, establishment at the array was normal, but maintenance after release of the tether failed, with significantly reduced H3K27me3 levels.

Together, these results establish RTT109 as a replication-linked factor essential for the mitotic stability of Polycomb-repressed chromatin, and they reveal a PRC1-independent route to epigenetic memory in fungi. The relevant substrate of RTT109 remains unknown. It may acetylate a histone residue other than lysine 56, transiently opening the H3 tail to facilitate methylation, or it may modify a non-histone protein; the Neurospora acetylome contains hundreds of acetylated proteins, including chromatin regulators such as the remodeler ISW and the deacetylase RPD3. Because RTT109 is fungal-specific and required for virulence in pathogens such as Candida albicans and Aspergillus fumigatus, the discovery that it underpins epigenetic plasticity also marks it as a potential therapeutic target. More broadly, the work adds to mounting evidence that the machinery of DNA replication is not merely a bystander in epigenetics but an active custodian of cellular memory.

Subject of Research: Epigenetic maintenance of PRC2-repressed chromatin by the histone acetyltransferase RTT109 in Neurospora crassa

Article Title: Epigenetic maintenance of PRC2-repressed chromatin requires RTT109 but not H3K56 acetylation

Article References: Epigenetic maintenance of PRC2-repressed chromatin requires RTT109 but not H3K56 acetylation. (n.d.). https://doi.org/10.1371/journal.pgen.1012327

Image Credits: AI Generated

DOI: 10.1371/journal.pgen.1012327

Keywords: epigenetics, PRC2, H3K27me3, RTT109, chromatin, Neurospora crassa, histone acetylation, facultative heterochromatin, DNA replication, gene silencing, epigenetic memory, PLOS Genetics