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Cell cycle dynamics regulate H3K27 and H3K9 histone modifications in Drosophila [1]

['Liyne Nogay', 'Faculty Of Biology', 'University Of Freiburg', 'Freiburg', 'International Max Planck Research School For Epigenetics', 'Biophysics', 'Metabolism', 'Ananthakrishnan Vijayakumar Maya', 'Lara Heckmann', 'Francesco Cardamone']

Date: 2026-03 

Cell cycle progression presents a fundamental challenge to epigenome integrity, particularly due to the need to reestablish post-translational histone modifications (PTMs) following DNA replication. Although proliferative and differentiating tissues exhibit markedly different cell cycle dynamics, how these differences shape the histone modification landscape in vivo remains largely unexplored. Here, we show that levels of H3K27ac, H3K27me3, and H3K9me3 are tightly linked to cell cycle dynamics in the Drosophila wing imaginal disc. We demonstrate that both physiological and pathological elongation of the cell cycle led to an accumulation of H3K9me3 and H3K27me3, whereas cell cycle acceleration reduces their levels. In contrast, H3K27ac exhibits the opposite pattern: levels decrease in arrested cells and increase with faster cycling. Genome-wide CUT&Tag analysis reveals that these changes predominantly affect genomic loci already modified in normally proliferating tissue. Importantly, the regulation of methylation levels at H3K9 and H3K27 is not solely mediated by the cell cycle machinery but reflects a metabolically guided process in which the rate of methylation is coupled to the rate of cell proliferation through metabolic activity, including signaling via the Insulin/PI3K/Akt pathway. Our study thus reveals key principles for understanding histone methylation in proliferating, senescent, and differentiating cells. In contrast, H3K27 acetylation is regulated through a distinct, cell cycle-coupled mechanism. We find that CBP/Nejire-mediated acetylation of H3K27 peaks during early and late S-phase and is reversed by HDAC1, as cells exit replication. Together, our findings establish a robust link between cell cycle progression and histone modification dynamics, highlighting the necessity of maintaining balanced PTM levels under varying proliferative states. These insights have broad implications for our understanding of development, aging, and tumor growth.

Funding: Funding for this work was provided by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (CIBSS – EXC-2189), the DFG Heisenberg Program to AKC (668189), as well as DFG grants to AKC (667603) and the Boehringer Ingelheim Foundation (BIF Plus3 & Rise Up) to AKC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All data, workflows, and FIJI based algorithms necessary to interpret the imaging data are included within the manuscript. The points extracted from images for analysis, the values used to build graphs, the values behind the means, and statistics reported can be found in S1 File (Source data and statistics). The code used for the mapping, normalization, peak calling, and signal quantification is available on Github: https://github.com/LaraH9/nogay_et_al_2025 . A version of record was uploaded to Zenodo: https://doi.org/10.5281/zenodo.18873410 . The CUT&Tag sequencing data generated in this study are provided on NCBI SRA: https://www.ncbi.nlm.nih.gov/sra/PRJNA1300380 .

Despite these insights, questions remain about how cell cycle progression influences histone modification dynamics, especially in in vivo settings. For instance, to what extent are histone modifications regulated in a cell cycle-dependent manner in developing tissues? How does the maintenance of these modifications differ between rapidly proliferating cells and cells in quiescent, senescent, or post-mitotic states? Moreover, how is the activity of histone-modifying enzymes coordinated with cell cycle checkpoints? To begin to address these questions we used the developing Drosophila wing imaginal disc to characterize cell cycle-dependent histone modifications and reveal pronounced dynamics of H3K9 and H3K27 modifications in a developing tissue.

Of note, strong acetylation of newly synthesized histones plays a specific cell cycle-dependent role during S-phase. Hyperacetylation, for example, at H4K5, H4K12, H3K14, H3K23, or H3K56, is observed during S-phase and facilitates recognition of newly synthesized histones by histone chaperone complexes, promoting their correct incorporation into newly synthesized chromatin. Hyperacetylation of histones presumably occurs in the cytoplasm and may be mediated by B-type acetyltransferases like Hat1 [ 49 – 59 ].

Importantly, the semi-conservative nature of DNA replication during S-phase has a profound impact on the landscape of these histone modifications. As the DNA is duplicated, pre-existing histones bearing PTMs are equally distributed onto the newly synthesized strands. Naïve, newly synthesized histones are integrated to ensure proper packaging of the duplicated genomes but also necessitating the reestablishment of the now diluted PTM code [ 11 42 – 44 ]. To reestablish the epigenetic histone modification landscape after incorporation of newly synthesized, naive histones, histone-modifying enzymes act post-replication by using the modifications on old recycled histones as a template to modify the newly incorporated histones [ 8 , 45 , 46 ]. Importantly, in cultured cells, histone acetylation marks are typically restored immediately after replication often via a transcription-dependent process, while histone methylation is generally reestablished during subsequent gap phases [ 9 , 10 , 47 , 48 ].

Some of the most abundant PTMs that regulate chromatin accessibility and transcriptional activity are acetylation and methylation of lysine residues on histone tails. While several lysine residues in histones can be acetylated or methylated, research has most strongly focused on dynamic acetylation and methylation of lysines 9 and 27 on histone H3 (H3K9 and H3K27), which represent some of the functionally most important histone modifications. In most species, trimethylation of H3K27 is mediated by Polycomb Repressive Complexes and relies on histone methyltransferases of the Enhancer of zeste E(z) family. H3K27me3 maintains the silencing of many genes required for cell fate specification during development, most famously Hox genes [ 24 – 28 ]. In contrast, trimethylation of H3K9 (H3K9me3) is mediated by the enzymatic activity of Su(var)3-9 and is central to the formation and maintenance of constitutive heterochromatin at telomeres, centromeres, and repetitive repeats [ 29 – 31 ]. In Drosophila, H3K27 acetylation is mediated by the histone acetyltransferase (HAT) Nejire (Nej), a homolog of mammalian p300/CBP, and is reversed by the histone deacetylase HDAC1/Rpd3 [ 32 – 35 ]. H3K27ac is strongly associated with active promoters and enhancers, where it is thought to facilitate chromatin opening and transcriptional activation [ 36 – 40 ]. As a consequence of these important functions, dysregulation of H3K9me3, H3K27me3, and H3K27ac is a common feature in cancer or other diseases characterized by alterations and defects in cell proliferation. Yet, importantly, it often remains unclear if problems with epigenetic modifications cause proliferation defects or if vice versa proliferation defects cause problems with epigenetic modifications [ 17 – 19 , 41 ]. Disentangling this relationship is key to understanding how epigenetic and proliferative states influence one another in development and disease.

In actively proliferating tissues, entry into the cell cycle at the G1/S transition, and progression through subsequent cycle phases, are tightly regulated. This regulation is mediated by Cyclins and Cyclin-dependent kinases (CDKs), as well as retinoblastoma (Rb) proteins and E2F transcription factors. Throughout the cell cycle, specific checkpoints respond to growth factors or cell size to modulate the rate of proliferation, as well as to DNA damage or chromosome misalignment to maintain the integrity of the genome [ 1 – 3 ]. As cells enter stages of differentiation, quiescence, or senescence, they typically withdraw from active cycling by either prolonging their G1 phase or entering a stable cell cycle arrest known as G0. This transition away from proliferation often involves CDK inhibitors, such as p21 and p27, which are intricately linked to gene expression programs that guide cell fate determination and differentiation [ 4 – 7 ]. Progression through the cell cycle presents significant challenges for chromatin organization and epigenetic regulation, especially during S-phase but also during mitosis. Specifically, during S-phase, chromatin must become highly accessible to facilitate replication fork progression, nucleosome redistribution and de novo nucleosome incorporation in the replicated genomes [ 7 – 12 ]. In contrast, in mitosis, chromatin must be tightly compacted to ensure accurate chromosome segregation [ 12 , 13 ]. Central to the regulation of chromatin organization are post-translational histone modifications (PTMs), which are also epigenetic regulators of transcriptional silencing and gene activation [ 14 – 16 ]. It is well-established that PTM dysregulation can lead to cell cycle defects by disrupting chromatin organization and entire gene regulatory networks [ 17 – 20 ]. While much attention has been dedicated to understanding how PTM dysregulation can drive pathogenesis by altering the expression of genes central to cell cycle regulation, less is known about how cell cycle dynamics connect to the maintenance and reestablishment of histone modifications. Yet, studies of the cell cycle-dependent regulation of the histone-modifying enzymes underscore the capacity of the cell cycle machinery to control specific histone modifications and thereby maintain chromatin function [ 21 – 23 ].

The regulation of the cell cycle and chromatin needs to be tightly coordinated to preserve a functional epigenome. Specifically, progression through S-phase poses several challenges: chromatin must become transiently accessible for DNA replication, pre-existing post-translationally modified histones must be evenly distributed between daughter genomes and newly incorporated histones must acquire appropriate modifications to compensate for the semi-conservative dilution of modifications. These demands are complicated by the fact that cell cycle progression and dynamics differ drastically across biological contexts, ranging from rapid divisions in embryonic and regenerating tissues to complete arrest in terminally differentiated cells. How chromatin states are maintained across diverse proliferative tissue environments in vivo is still insufficiently understood.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003371

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