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

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.

Fig. 1

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.

Fig. 2

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.

Fig. 3

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.