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. 2025 Dec 2;49(1):100303. doi: 10.1016/j.mocell.2025.100303

Set1-dependent H3K4 methylation is essential for sustained gene expression at newly activated loci

Shinae Park 1, Kyungmin Lee 1, Junsoo Oh 1, Jung-Shin Lee 1,⁎
PMCID: PMC12804360  PMID: 41344592

Abstract

Histone H3 lysine 4 trimethylation (H3K4me3) has been associated with active transcription, yet whether it plays a causative role in gene activation remains an open question. In this study, we reveal that the deletion of Paf1 complex subunit Leo1 in Saccharomyces cerevisiae induces robust transcriptional activation at a subset of genes, particularly those involved in sterol transport, without altering global H3K4me3 levels. These induced genes acquire de novo H3K4me3 at promoter-proximal regions, and this transcriptional induction is entirely dependent on Set1, the sole methyltransferase responsible for H3K4me3. Strikingly, loss of Set1 abolishes expression of these genes, even in the presence of previously established H3K4me3, and their expression is fully restored upon Set1 reintroduction. These effects are specific to Leo1 deficiency and not observed in other Paf1C mutants. Furthermore, Set1-dependent gene activation enhances sterol uptake, underscoring its physiological relevance. Our findings provide direct in vivo evidence that Set1-catalyzed H3K4me3 is not merely a transcriptional correlate, but a context-dependent driver of gene expression.

Keywords: Gene expression, H3K4 methylation, Leo1, Set1

INTRODUCTION

Chromatin structure plays a central role in regulating gene expression through post-translational modifications of histones. These modifications regulate chromatin accessibility by altering the overall charge of histone tails or by providing specific binding sites for transcriptional regulators (Bannister and Kouzarides, 2011). Among these, methylation of histone H3 at lysine 4 is one of the most extensively studied. Monomethylation, dimethylation, and trimethylation of H3K4 (H3K4me1, H3K4me2, and H3K4me3, respectively) are catalyzed by the Set1 complex (Dehe and Geli, 2006, Deshpande and Bryk, 2024, Shilatifard, 2006). High-throughput techniques have revealed that H3K4me3 is highly enriched at promoter-proximal regions of actively transcribed genes (Oh et al., 2024, Park et al., 2020, Santos-Rosa et al., 2002), and it is widely used as a chromatin mark of transcriptional activity across eukaryotes.

Despite this association, the functional significance of H3K4me3 remains highly debated. Previous studies indicate that H3K4me3 is involved in modulating RNA polymerase II pause-release and transcriptional elongation than in directly initiating transcription (Wang et al., 2023). Moreover, global perturbation of H3K4me3 levels, either through depletion or enhancement, has minimal impact on transcriptional output (Howe et al., 2017). For instance, deletion of Spp1 in yeast, which impairs Set1 recruitment, reduces H3K4me3 but has no measurable effect on mRNA levels, and loss of the demethylase Jhd2 leads to increased H3K4me3 without enhancing transcription (Lenstra et al., 2011, Liang et al., 2007, Margaritis et al., 2012). These findings have supported the notion that H3K4me3 may act more as a transcriptional footprint than as a causal regulator.

However, emerging evidence supports an instructive role for H3K4me3 under certain conditions. In mammalian cells, H3K4me3 engages the TFIID subunit TATA-box binding protein associated factor 3 (TAF3) to facilitate transcription initiation at p53 target genes (Lauberth et al., 2013). Epigenome editing approaches have further demonstrated that artificial installation of H3K4me3 can trigger transcription by remodeling the chromatin environment in a hierarchical manner (Policarpi et al., 2024). Targeted induction of H3K4me3 has been shown to overcome epigenetic silencing in DNA methylation status (Cano-Rodriguez et al., 2016). Yet, despite these advances, no study has directly demonstrated that the complete loss of Set1—and consequently H3K4 methylation—leads to the failure of gene expression at loci where H3K4me3 had previously been acquired.

In this study, we sought to define conditions under which Set1-mediated H3K4 methylation becomes functionally indispensable for gene expression. Using Saccharomyces cerevisiae as a model, we identified a set of genes that become transcriptionally activated and acquire promoter-proximal H3K4me3 de novo. We show that continued expression of these genes strictly depends on Set1, establishing H3K4 methylation is not merely a mark of post transcriptional activity, but a prerequisite for sustained gene expression at newly activated loci.

MATERIALS AND METHODS

Yeast Strains and Culture Conditions

The strains used in this study are presented in Table S2. For RNA-seq, qRT-PCR, and western blot analyses, cells were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30°C and harvested at an optical density at 600 nm of 1.0. For anaerobic conditions, cultures were incubated in tightly sealed screw-cap tubes filled to the brim with YPD medium and placed in a BD GasPak EZ anaerobic pouch system (BD, 260683).

Western blot analysis

Western blot assay was performed as previously described (Lee et al., 2007). The harvested cells were homogenized by vortexing at 4°C for 30 minutes. The resulting lysates were then boiled with loading dye to denature the proteins and prepare them for analysis by western blotting. The following antibodies were used in western blot: anti-H3, anti-H3K4me1, anti-H3K4me2 (obtained from Ali Shilatifard's Laboratory), and anti-H3K4me3 (Merck, 07-473).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were conducted following a previously established protocol (Lee et al., 2007). Briefly, yeast cells were grown to the exponential phase (OD600 of 1.0) at 30°C. After cross-linking with formaldehyde for 5 minutes and quenching with 2.5 M glycine for 20 minutes, the cells were harvested. For cell lysis, the cell pellet was resuspended in FA-lysis buffer, and 0.5 mm glass beads were added. The cells with beads were subjected to bead beating at 4°C for 30 minutes. The chromatin of the lysed cells was then sheared by sonication. Immunoprecipitation was performed using antibodies specific to H3K4me3 (Merck, 07-473). To normalize the ChIP experiments, 10% of the chromatin extract from Saccharomyces pombe was added as a spike-in control. A/G agarose (Santa Cruz Biotechnology, sc-2003) was used to precipitate the DNA. Following the washing steps, reverse cross-linking and DNA precipitation were performed to obtain the ChIP-enriched DNA fragments.

ChIP-Seq and Data Analysis

The ChIP-seq DNA samples were quantified using the Quant-iT PicoGreen dsDNA Assay kit, and a starting material of 10 ng of DNA was used for library preparation. For sequencing, library construction was performed using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB, E6240). Sequencing was carried out on an Illumina HiSeq 2500 instrument, following the manufacturer's protocol.

Sequencing adapters were removed, and quality-based trimming was performed using Trimmomatic v0.36 (Bolger et al., 2014). The resulting cleaned reads were aligned to the reference genome using bowtie2 v2.2.5 with default parameters (Langmead and Salzberg, 2012). To normalize the data, the reads specific to S. pombe were calculated and adjusted to one million reads by the spike-in normalization method. Visualization of the data was performed using EaSeq and the IGV (Brachmann et al., 1998; Lerdrup et al., 2016). A total of 6,020 protein-coding genes were selected from the sacCer3 reference genome, excluding noncoding exons from the initial set of 7,589 genes. The mean reads per million values were calculated for each gene, and heatmaps and anchor plots were generated using EaSeq (Lerdrup et al., 2016).

RNA Extraction and Quantitative RT-PCR Analyses

Total RNA was isolated using NucleoSpin RNA (MN, MN740955) according to the manufacturer’s protocol. For quantitative RT-PCR, 1 µg of RNA was used for cDNA synthesis. The cDNA synthesis was performed using cDNA synthesis kit (Enzynomics, EZ005S) according to the manufacturer’s protocol. The sequences of primers used for amplification are presented in Table S3.

RNA-Seq and Data Analysis

mRNA isolation was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). Subsequently, a strand-specific sequencing library was synthesized following the protocol of the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, E7420). Analysis of differentially expressed genes was performed with DEseq2 normalization on R Studio. Expression data were visualized using a volcano plot generated by ggplot2 ggrepel R packages (Slowikowski, 2024; Wickham, 2016) and Integrative Genomics Viewer (IGV) genome browser (Robinson et al., 2011). Heatmap was generated by Pheatmap R package. Volcano plot was visualized using SRplot (Tang et al., 2023).

NBD-Cholesterol Uptake Assay

Sterol uptake assay was performed as previously described (South et al., 2013). Cells were incubated with 20 µg/ml 22-NBD-cholesterol (Invitrogen, N1148) for overnight (16 hours). Overnight cultured cells were diluted to OD600 of 1.0 and harvested. After washing 2 times, cells were observed under a microscope.

RESULTS

Set1-Mediated H3K4me3 is Elevated at Newly Activated Promoters in Leo1-Deficient Cells

To understand the conditions under which H3K4me3 plays a functionally instructive role in transcription, we sought a chromatin context where global levels of H3K4 methylation remain intact, but transcriptional changes occur at defined loci. Deletion of Leo1, a core component of Paf1 complex (Paf1C), offered such a context. Notably, Leo1-deficient cells exhibit a gene expression profile that is distinct from deletion strains of other Paf1C components. At the same time, global levels of H3K4 methylations remain largely unaltered in Leo1-deficient cells, providing a rare opportunity to dissect locus-specific contributions of H3K4me3 without confounding global disruption. We therefore hypothesized that such a background could unmask functional requirements for H3K4me3 at newly induced loci.

To assess genome-wide H3K4 methylation in this setting, we performed western blot analysis for H3K4me1, H3K4me2, and H3K4me3 in wild-type and leo1Δ strains (Fig. 1a). As controls, we included a set1Δ strain, which lacks the sole H3K4 methyltransferase, and a bre1Δ strain, which lacks the E3 ligase required for H2Bub, a prerequisite for H3K4me2 and H3K4me3. As expected, all 3 methylation states were completely absent in the set1Δ strain and substantially reduced in bre1Δ. In contrast, leo1Δ cells retained near wild-type levels of all 3 marks, confirming that global H3K4 methylation is preserved in the absence of Leo1.

Fig. 1.

Fig. 1

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A subset of upregulated genes in leo1Δ cells acquires de novo promoter-proximal H3K4me3. (a) Western blot shows intact all 3 H3K4 methylation states (H3K4me1, H3K4me2, and H3K4me3) in leo1Δ cells. (b) Metagene plots show average H3K4me1, H3K4me2, and H3K4me3 signal around 1,500 bp from transcription start sites. (c) Volcano plot showing differentially expressed genes between WT (control) and leo1Δ cells. Orange dots indicate genes significantly upregulated in leo1Δ (n = 71), sky blue dots represent significantly downregulated genes (n = 150), and gray dots indicate genes with no significant change in expression. Value of X-axis is log2 fold change leo1Δ/WT and value of Y-axis is −log10(Padj). (d) Schematic illustration displays how H3K4me3 enrichment in leo1Δ cells was quantified relative to wild-type in this study. (e) Venn diagram shows the number of upregulated genes having increased H3K4me3 enrichment around 100 bp from TSSs. (f) Metagene plots display the average H3K4me1 (left), H3K4me2 (middle), and H3K4me3 (right) signals across the 28 upregulated genes with increased H3K4me3. (g) Schematic illustration outlining how de novo H3K4me3 peaks were defined and how the 8 genes were selected. (h) Integrative Genomics Viewer (IGV) tracks display the profile of H3K4me3 and RNA-seq at the 8 genes gained de novo H3K4me3 in leo1Δ cells. TSS, transcription start site.

We next used ChIP followed by sequencing (ChIP-seq) to examine whether the genome-wide distribution of H3K4 methylation states was altered. In both wild-type and leo1Δ strains, H3K4me3 was sharply enriched at transcription start sites (TSSs), H3K4me2 extended across gene bodies, and H3K4me1 localized toward the 3′ ends (Fig. 1b, Fig. S1a). Thus, Leo1 deletion preserves canonical patterns of H3K4 methylation, providing a sensitized background to study gene-specific effects of H3K4me3.

The preservation of canonical H3K4 methylation patterns in leo1Δ cells provides a unique opportunity to investigate the locus-specific function of H3K4me3 without global disruption. To determine whether gene expression changes correlate with local gains in H3K4me3, we performed RNA-seq in wild-type and leo1Δ strains. We identified 71 genes significantly upregulated and 150 downregulated in the absence of Leo1 (Fig. 1c).

We focused on the upregulated set and assessed H3K4me3 enrichment at promoter-proximal regions (−100 to +100 bp relative to the TSS), a zone known to harbor transcriptionally relevant histone modifications including H3K4me3 (Oh et al., 2024, Pokholok et al., 2005, Soares et al., 2017, Wang et al., 2023, Zhu et al., 2024). We quantified H3K4me3 signal intensity across this window and identified genes with a leo1Δ-to-WT H3K4me3 signal ratio greater than 1 as having increased promoter-proximal H3K4me3 (Fig. 1d). This analysis showed that 1,462 genes exhibited elevated promoter-proximal H3K4me3 in leo1Δ cells. Among the 71 upregulated genes, 28 also showed increased H3K4me3 signal in promoter-proximal regions in leo1Δ relative to wild-type (Fig. 1e and Table S1). This subset suggests a coordinated gain in H3K4me3 and transcription, raising the possibility that H3K4me3 plays a direct role in their activation.

Average methylation profiles across these 28 genes confirmed that promoter-proximal H3K4me3 levels were markedly elevated in leo1Δ cells compared to WT (Fig. 1f). H3K4me2 levels were similarly elevated and extended to the gene body, whereas H3K4me1 showed no appreciable change. Closer inspection of ChIP-seq peak profiles revealed that 8 of the 28 genes acquired de novo H3K4me3 peaks at promoter-proximal regions in leo1Δ cells that were undetectable in wild-type (Fig. 1g, h). The remaining 20 genes either showed moderate peak intensity changes or peak shifts likely originating from adjacent promoters (Fig. S1c and d). The remaining 43 upregulated genes showed no increase in H3K4me3 or fell below the enrichment threshold (Fig. S1e).

These data indicate that Set1-mediated H3K4me3 is locally and specifically elevated at a subset of genes that become transcriptionally activated in the absence of Leo1, revealing a chromatin context in which H3K4me3 may function as a necessary driver of gene expression.

Set1-Dependent H3K4me3 is Required for Transcriptional Activation of De Novo Marked Genes in Leo1-Deficient Cells

To determine whether genes acquiring de novo H3K4me3 peaks in leo1Δ are dependent on this modification for expression, we generated a leo1Δset1Δ double mutant and compared its transcriptome with that of the leo1Δ single mutant. Among the 202 genes that were downregulated more than two-fold in leo1Δset1Δ relative to leo1Δ, only 13 overlapped with both the 71 genes upregulated in leo1Δ compared to wild-type and the 1,462 genes showing increased promoter-proximal H3K4me3 in leo1Δ cells (Fig. 2a). Remarkably, all eight genes previously identified as acquiring de novo H3K4me3 and showing strong transcriptional upregulation in leo1Δ were among those downregulated in the double mutant (Figs. 1 and 2a, b). These findings provide direct evidence that Set1-mediated H3K4me3 is required for activation of this subset of genes in the absence of Leo1.

Fig. 2.

Fig. 2

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Expression and function of de novo H3K4me3-marked genes in leo1Δ cells depend on Set1. (a) Venn diagram shows the overlap among genes that are upregulated in leo1Δ cells relative to wild-type, downregulated in leo1Δset1Δ compared to leo1Δ, and exhibit increased H3K4me3 enrichment in leo1Δ cells. (b) Bar graph shows normalized expression counts for the 8 genes with de novo H3K4me3 peaks in WT, leo1Δ, and leo1Δset1Δ cells. (c) Descriptions of the functions of the 8 genes, based on the Saccharomyces Genome Database (https://www.yeastgenome.org/). (d-f) RT-PCR results show the expression of 3 representative genes ((d) DAN4, (e) HES1, and (f) PBI1) among the 8 with de novo H3K4me3 in WT, leo1Δ, set1Δ, and leo1Δset1Δ cells under normoxic and hypoxic conditions. Student's t-test was used to compared the groups. One, 2, or 3 asterisks (*, **, ***) are used to indicate the level of statistical significance, corresponding to P-values of ≤.05, ≤.01, and ≤.001, respectively. n.s. means not significant. (g) Quantification of sterol uptake using NBD-cholesterol fluorescence in WT, leo1Δ, and leo1Δ set1Δ was performed by adding NBD-cholesterol to the culture medium. Scale bar represents 100 µm. (h) Western blot shows restoration of H3K4me3 by reintroducing pRS315-SET1 in leo1Δset1Δ cells.

Interestingly, 8 genes that gained de novo H3K4me3 in leo1Δ cells—PBI1, TIR1, TIR3, HES1, DAN4, YMR317W, SET4, and DAN1—are known transcriptional targets of Upc2, a sterol-responsive transcription factor activated under hypoxic or sterol-depleted conditions (Jorda and Puig, 2020, Wilcox et al., 2002). To assess the physiological relevance of this mechanism, we examined the expression of these genes under hypoxic conditions. Thus, we hypothesized that hypoxia would further enhance the expression of these genes in leo1Δ cells, but not in the absence of Set1. Using an anaerobic pouch system, we cultured cells under aerobic and hypoxic conditions and performed RT-PCR. As expected, DAN4 expression was strongly induced by hypoxia in wild-type cells and showed an even greater induction in leo1Δ cells (Fig. 2d).

Notably, the increased expression of these H3K4me3-marked genes was abolished in leo1Δset1Δ cells, in which transcript levels reverted to wild-type levels. These results demonstrate that Set1-dependent H3K4me3 is essential for the transcriptional activation of de novo H3K4me3-marked genes in leo1Δ cells, and further suggest that this histone modification may prime promoters for rapid transcriptional responses to environmental cues (Wang and Helin, 2025).

Under hypoxic conditions, the DAN/TIR family replaces aerobic mannoproteins in the cell wall and facilitates sterol uptake (Abe, 2007). To determine whether this transcriptional program has a physiological outcome, we assessed sterol uptake using fluorescent NBD-cholesterol. Consistent with enhanced expression of DAN/TIR family genes, leo1Δ cells showed increased sterol uptake (Figs. 2g and S2a). This phenotype was lost in the leo1Δset1Δ mutant but was fully restored upon SET1 reintroduction (Fig. 2h), indicating that Set1-dependent transcription of de novo H3K4me3-marked genes directly contributes to sterol acquisition.

To determine whether the H3K4me3-marked, upregulated genes in leo1Δ cells depend on Set1/COMPASS, we examined Set1 occupancy by performing ChIP-qPCR on 2 representative H3K4me3-acquired genes (TIR1 and PBI1) using an anti-Set1 antibody in WT, leo1Δ, and set1Δ (negative control) strains. Set1 recruitment at their promoter regions is markedly increased in the leo1Δ strain, corresponding with the acquisition of de novo H3K4me3 (Fig. S2b). In contrast, the housekeeping gene ACT1 showed no difference in Set1 occupancy between WT and leo1Δ. These findings indicate that LEO1 deletion specifically enhances Set1/COMPASS recruitment at newly activated loci.

Activation of De Novo H3K4me3-Marked Genes Is Specific to Leo1 Deletion and Not a General Consequence of Paf1C Disruption

To determine whether the gene expression changes observed in leo1Δ cells reflect a general consequence of Paf1 complex (Paf1C) disruption or are specific to the loss of Leo1, we performed RNA-seq in paf1Δ cells and compared the expression profiles of the 71 upregulated and 150 downregulated genes previously identified in leo1Δ cells (Fig. S3a-d). Among the genes upregulated in leo1Δ, only 8 were also increased in paf1Δ, while 5 were actually downregulated. Strikingly, TIR1—one of the most strongly induced genes in leo1Δ—was downregulated more than 2-fold in paf1Δ cells (Figs. 1 and S3e). Although DAN1 was upregulated in both mutants, the magnitude of induction was substantially greater in leo1Δ (log₂ fold change > 3.6) than in paf1Δ (log₂ fold change > 1.1).

To test whether this transcriptional response is unique to Leo1 loss, we assessed the expression of TIR1 and DAN1 in deletion mutants of all Paf1C subunits by RT-PCR. Only leo1Δ cells exhibited a pronounced increase in expression of both genes (Fig. S3f). These findings indicate that the induction of genes with de novo H3K4me3 is not a general consequence of Paf1C dysfunction but is specific to the loss of Leo1.

Consistent with this, the physiological phenotype associated with the activation of DAN/TIR genes was also specific to leo1Δ. NBD-cholesterol uptake assays showed increased sterol incorporation in leo1Δ cells, whereas deletion of other Paf1C subunits failed to reproduce this effect (Fig. S3g).

In contrast, among the 150 genes downregulated in leo1Δ, 46—including phosphate metabolism genes such as PHO84, PHO89, and SPL2—were also downregulated in paf1Δ (Fig. S3h and i). These results suggest that transcriptional repression of phosphate-related genes reflects a general requirement for Paf1C integrity, in agreement with previous studies (Ellison et al., 2019).

Although the effects we describe emerge in the absence of Leo1, further mechanistic dissection will be needed to clarify how this subunit influences transcriptional regulation and chromatin architecture. Nonetheless, our findings position Leo1 deletion as a distinctive chromatin perturbation—one that unmasks a functional requirement for Set1-mediated H3K4 methylation at specific promoters. This study adds to growing evidence that H3K4me3 is not merely a passive mark of transcriptional activity, but under defined chromatin contexts, can act as an active and necessary driver of gene expression.

DISCUSSION

Although H3K4me3 has long been associated with active promoters, its role as a causal driver of transcription remains controversial. Here, we provide in vivo evidence that Set1-mediated H3K4me3 is essential, not merely correlative, for sustained transcription at a subset of newly activated loci particularly those involved in sterol uptake, in the absence of Leo1. In this context, sterols are efficiently imported from the extracellular environment, yet transcription at these loci collapses upon SET1 deletion and resumes upon its reintroduction, despite prior H3K4me3 enrichment. This demonstrates that continuous Set1 methyltransferase activity is required to maintain gene expression.

We next asked how the loss of Leo1 enables these loci to acquire de novo H3K4me3. Our data further indicate that Leo1 does not act directly at these target promoters (Fig. S3j). Published ChIP-exo analysis (Rossi et al., 2021) demonstrated that Leo1 is not recruited to the 8 genes that acquire de novo H3K4me3 in the leo1Δ background, consistent with their low basal transcription in wild-type cells. Instead, Leo1 associates with the transcription factor UPC2, a master regulator of sterol uptake and membrane remodeling.

We therefore propose a model for how Leo1 influences sterol-responsive transcription (Fig. 3). Loss of Leo1 may permit Upc2-dependent activation of these genes, after which Set1 is required to establish and maintain H3K4me3 at their promoters. In this model, Leo1 normally constrains sterol uptake gene expression not by blocking Set1 recruitment per se, but by restricting transcription factor–driven promoter activation. This regulatory architecture explains why only the leo1Δ background, and not other Paf1C mutants, reveals robust de novo H3K4me3 formation and Set1 dependency.

Fig. 3.

Fig. 3

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Context-dependent requirement for Set1-mediated H3K4 trimethylation in gene activation in the absence of Leo1. (a) In wild-type cells, Leo1 is not recruited to the promoters of the sterol uptake genes and these genes remain transcriptionally silent under normoxic conditions. However, Leo1 associates with the transcription factor Upc2, which regulates sterol metabolism genes. (b) We propose that deletion of Leo1 leads to derepression of sterol uptake genes. Under these conditions, Set1-mediated H3K4 trimethylation is deposited de novo at these loci and becomes essential for sustaining transcriptional activation. (c) In contrast, in the absence of Set1, the sterol uptake genes fail to maintain expression, demonstrating a context-dependent requirement for ongoing H3K4 methylation. Importantly, other Paf1C components do not show this phenotype, indicating that the regulatory function of Leo1 in sterol metabolism is distinct from general Paf1C activity.

Leo1 deletion appears to create a permissive chromatin environment that reveals a specific Set1 dependency. While other Paf1C components broadly impact transcription and chromatin state (Ellison et al., 2019), Leo1 loss leads to activation of a defined group of genes—especially those involved in sterol uptake—that acquire de novo H3K4me3. These loci remain silent in other Paf1C mutants, underscoring the specificity of the Leo1 effect.

Our findings align with prior studies where artificial H3K4me3 deposition relieved gene silencing in a context-dependent manner (Cano-Rodriguez et al., 2016, Policarpi et al., 2024). Extending this concept, we show that endogenous Set1 activity is required to sustain transcription in a chromatin context uniquely revealed by Leo1 loss.

Notably, while we observed H3K4me3 gain and transcriptional activation in yeast, Laurent et al. (2023) reported H3K9me3 enrichment and gene repression upon LEO1 loss in human fibroblasts (Laurent et al., 2023). These contrasting outcomes suggest that Leo1 may help balance active and repressive chromatin states in a species- or context-dependent manner.

Finally, while our study identifies a chromatin setting in which H3K4me3 becomes functionally indispensable, the precise role of Leo1 remains unclear. Whether Leo1 directly regulates transcription factor activity, chromatin structure, or cotranscriptional histone modification dynamics is unknown. In particular, the possible involvement of Upc2 or chromatin remodeling complexes in mediating Set1 dependency warrants further investigation.

Together, our results establish that H3K4me3 can function as an instructive mark for transcription, depending on the chromatin environment. The Leo1-deficient background offers a useful model for dissecting the mechanisms by which histone modifications influence transcriptional outcomes beyond mere association with activity.

Author Contributions

Kyungmin Lee: Investigation. Shinae Park: Writing – review & editing, Writing – original draft, Investigation. Junsoo Oh: Writing – review & editing. Jung-Shin Lee: Writing – review & editing, Writing – original draft, Supervision, Investigation, Conceptualization.

Declaration of Competing Interests

The authors declare no competing interests.

Acknowledgments

We thank Ali Shilatifard for providing strains and anti-H3, anti-H3K4me1, and anti-H3K4me2 antibodies. This research was supported by the National Research Foundation of Korea (NRF) and the Institute of Information & Communications Technology Planning & Evaluation grants funded by the Korea Government (Ministry of Science and ICT and Ministry of Education) (No. NRF-2023R1A2C1003171, RS-2025-25396383 and IITP-2025-RS-2023-00260267 to J.L., NRF-2021R1C1C2005724 to S.P.). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2024-00461215 to J.L. and S.P.).

Footnotes

Appendix A

Supplemental material associated with this article can be found online at doi:10.1016/j.mocell.2025.100303.

Appendix A. Supplemental material

Supplementary material

mmc1.pdf (5.1MB, pdf)

.

Data Availability Statement

The ChIP-seq and RNA-seq data discussed in this study have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE303595 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303595) and GSE303407 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303407).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material

mmc1.pdf (5.1MB, pdf)

Data Availability Statement

The ChIP-seq and RNA-seq data discussed in this study have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE303595 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303595) and GSE303407 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303407).

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