The structural organization of eukaryotic DNA into chromatin regulates transcription by RNA polymerases (Han and Grunstein 1988, Izban and Luse 1992, Kornberg and Thonmas 1974, Wasylyk and Chambon 1979, Workman and Kingston 1998). Nucleosomes, the fundamental structural subunits of eukaryotic chromatin, consist of 147 bp of double-stranded DNA wound around a histone core octamer (Lawrence, et al. 2016, Luger, et al. 1997, Venkatesh and Workman 2015). The histone core octamer has two copies of each of the histones, H2A, H2B, H3 and H4. To overcome the physical barrier imposed by chromatin, RNA polymerases rely on the action of trans-acting proteins and protein complexes, including chromatin remodelers, transcription factors, co-activators and histone-modifying enzymes make chromatin accessible to RNA polymerase II to access DNA sequences (Castillo, et al. 2017, Chatterjee, et al. 2011, Côté, et al. 1998, Lee, et al. 1993, Lee, et al. 2007, Santos-Rosa, et al. 2003).
Post-translationally modified histones regulate chromatin characteristics and functions, including accessibility, compaction, nucleosome dynamics, replication, and transcription (Chatterjee, et al. 2011, Lawrence, et al. 2016, Santos-Rosa, et al. 2003, Soriano, et al. 2014, Zhang, et al. 2015). Histones are targets for many covalent modifications including methylation, acetylation, phosphorylation and ubiquitylation (Allfrey, et al. 1964, Zhang, et al. 2015). Covalently modified histones in chromatin may recruit or facilitate the interaction of effectors, such as chromatin-remodeling complexes and transcription factors, with chromatin (Castillo, et al. 2017, Lawrence, et al. 2016). For example, acetylated lysines are recognized by bromodomain-containing proteins ((Dhalluin, et al. 1999); reviewed in (Filippakopoulos and Knapp 2014)), like the Swi2/Snf2 subunit in the SWI/SNF chromatin-remodeling complex, which aids in the generation of open and accessible chromatin ((Tamkun, et al. 1992), reviewed in (Swygert and Peterson 2014)). The dynamic exchange of acetyl groups onto and off of histones acts as a switch between active and repressive chromatin, respectively (reviewed in (Eberharter and Becker 2002, Kuo and Allis 1998)). In contrast to histone acetylation, the outcome of histone methylation is context-dependent; methylated histones have the capacity to activate or repress transcription (Hyun, et al. 2017).
Methylation of histones is catalyzed by histone methyltransferases (HMTases). Set1 is an HMTase that catalyzes the mono-, di- and trimethylation of the fourth lysine on the amino terminal tail of histone H3 (Briggs, et al. 2001, Qu, et al. 2018, Shilatifard 2012). S. cerevisiae Set1 is a member of the COMPASS complex (Complex Associated with Set1, Figure 1) (Bae, et al. 2020, Briggs, et al. 2001, Miller, et al. 2001, Morillon, et al. 2005, Mueller, et al. 2006, Roguev, et al. 2001). The function of members of the SET family of H3K4 HMTases are conserved in eukaryotes (Miller, et al. 2001, Takahashi, et al. 2011). Mutations in Set1-like H3K4 HMTases alter segmentation in Drosophila melanogaster and floral development in Arabidopsis thaliana (Breen 1999, Jiang, et al. 2011, Shilatifard 2012). The human homologs of Set1, including MLL1 and its translocation alleles, are implicated in several hematological malignancies, such as mixed lineage leukemia, acute myeloid leukemia and acute lymphoblastic leukemia (Kandoth, et al. 2013, Roguev, et al. 2001, Ruault, et al. 2002, Shilatifard 2012, Slany 2009). The conservation of Set1-like proteins from yeast to humans underscores their importance in biological processes.
The effect of H3K4 methylation on chromatin accessibility depends on the chromatin context, consistent with data showing that H3K4 mono-, di-, and trimethylation may have different effects on gene transcription (Kusch 2012, Pokholok, et al. 2005). Chromatin immunoprecipitation (ChIP) and ChIP-seq experiments revealed that the distribution of K4-monomethylated (H3K4me1), K4-dimethylated (H3K4me2) and K4-trimethylated (H3K4me3) histone H3 across open reading frames (ORFs) is not identical (Bernstein, et al. 2005, Soares, et al. 2017). For the most highly expressed genes in S. cerevisiae, nucleosomes with H3K4me3 peak at the promoter and up to ~200 bp beyond the transcription start site, nucleosomes with H3K4me2 are enriched in the middle of the ORF, and nucleosomes with H3K4me1 are found predominantly at the 3’ end of an ORF (Berger 2007, Pokholok, et al. 2005). Methylation of histones is not known to change the structure of chromatin on its own, instead methylated histones act by recruiting effector proteins to chromatin (Musselman, et al. 2012, Pray-Grant, et al. 2005, Taverna, et al. 2006).
H3K4-methylated histones may be recognized by chromodomain-containing proteins (Eissenberg 2012) and plant homeodomain (PHD) finger domains, including the COMPASS member Spp1 (He, et al. 2019). H3K4me3 is usually associated with active transcription (Kusch 2012, Ng, et al. 2003, Schneider, et al. 2005). H3K4me3 is recognized by the chromodomain-containing protein, Chd1, a member of the SAGA transcription coactivator complex (Pray-Grant, et al. 2005). The transcription factor TAF3 also interacts with trimethylated H3K4 to recruit TFIID to gene promoters (Vermeulen, et al. 2007). In yeast, the chromatin remodeler Isw1 interacts with methylated H3K4 to generate open, accessible chromatin at the 5’ end of the MET16 gene (Santos-Rosa, et al. 2003). H3K4me3 is also recognized by other protein complexes, some of which are negative effectors of transcription (Musselman, et al. 2012, Taverna, et al. 2006). In response to DNA damage, the PHD domain of ING2 (INhibitor of Growth 2), a subunit of the mSin3a–HDAC1 histone deacetylase complex, binds to H3K4me3, stabilizing mSin3a–HDAC1 at the promoter of cyclin D1 gene and other proliferation genes, leading to repression of transcription (Shi, et al. 2006). H3K4me2 is associated with repression of transcription and has been shown to interact with HDACs that reduce acetylated histones at the 5′ ends of some highly expressed genes (Kim and Buratowski 2009, Pinskaya and Morillon 2009).
Progress is being made toward understanding the roles H3K4me1 plays in gene regulation. In yeast, H3K4me1 regulates chromatin remodeling at osmostress-induced genes (Nadal-Ribelles, et al. 2015). In mammalian cells, there is compelling evidence that H3K4me1 promotes interactions between enhancers and promoters by facilitating the binding of chromatin remodelers (Local, et al. 2018, Yan, et al. 2018). However, a catalytically defective H3K4 HMTase also facilitates enhancer-promoter interactions meaning that an additional mechanism that doesn’t require H3K4me1 is likely to promote enhancer-mediated effects as well (Dorighi, et al. 2017, Rickels, et al. 2017). Recent work has shown that the patterns of H3K4me1 with H3K4me3 and H3K27me3 at promoters in human and mouse germ cells and ESCs may predict the transcriptional state of a promoter (Bae and Lesch 2020).
Set1 is the only H3K4 HMTase in S. cerevisiae making it an excellent system to study the effect of the three H3K4 methyl marks on transcription. In a previous study, mutants of SET1 were made that encode proteins with amino acid substitutions in the SET domain of Set1 (Figure 1) (Williamson, et al. 2013). Wild-type Set1 generates H3K4me1, H3K4me2 and H3K4me3. Amino acid substitution mutants were constructed to alter residues near and in the active site of Set1 to generate mutants with different H3K4 methylation capabilities. The set1-Y967A alleles is null mutant with no detectable H3K4 methylation that is indistinguishable from a set1D mutant. The set1-G951A mutant produces H3K4me1 and very low levels of H3K4me2. Although G951 is not in the active site, it is highly conserved in Set1 homologs and has been shown to be important for Set1 function (Dillon, et al. 2005, Lee, et al. 2018, Nislow, et al. 1997, Sollier, et al. 2004). Two additional partial function mutants were generated: set1-Y967F that generates H3K4me1 and low levels of H3K4me3 and set1-R1013H that generates H3K4me1 and H3K4me2 (Figure 1).
In this study, the set1 amino-acid substitution mutants and the HIS3 gene were studied to learn about the roles of individual H3K4 methyl marks in RNA polymerase II transcription in S. cerevisiae. HIS3 codes for imidazole glycerol phosphate dehydratase (Fink 1964), the enzyme that catalyzes the sixth step in the biosynthesis of histidine in S. cerevisiae. The herbicide 3-Amino-1,2,4-triazole (3AT) is a competitive inhibitor of the HIS3 gene product and is used to induce histidine-starvation leading to the production of the general amino acid control regulator, Gcn4 (Brennan and Struhl 1980, Hope and Struhl 1985). Gcn4 activates transcription of the HIS3 gene, increasing the level of HIS3 mRNA and biosynthesis of histidine (Hill, et al. 1986).
Our work shows that in the absence of H3K4me2 and H3K4me3, H3K4me1 promotes transcription of the HIS3 gene under histidine-starvation conditions in S. cerevisiae. A role of H3K4me1 in activation of transcription has not been reported previously. In addition, we also demonstrate that one or more of the genes required for biosynthesis of isoleucine and valine (Falco and Dumas 1985, Falco, et al. 1985) is activated by H3K4me1 when H3K4me3 and H3K4me2 are absent. The results indicate a previously unrecognized role for H3K4me1 in the activation of transcription of the HIS3 gene and one or more of the ILV genes by RNA polymerase II.