dSet1 is the main H3K4 di- and tri-methyltransferase throughout Drosophila development

Abstract

In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a "global" or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans.

Figure 1

The lethal 5 locus encodes the Drosophila ortholog of the Set1 H3K4 methylase (dSet1). (A) Using single-embryo PCR mapping, we demonstrate that CG40351 is absent in deficiencies that remove the lethal 5 and lethal 4B loci. (Top row gel lanes) CG40351 amplicons. (Bottom row gel lanes) Grip84 amplicons (+ve control). (B) Transcript model of dSet1/CG40351-RB. The position of the start codon (ATG) and stop codon (TAA) are indicated with small arrows. Locations of mutations are given below relative to the translational start site and are marked by large arrows along the gene model. Descriptions of mutations identified are the following: mutant G12 has a deletion of nt A1717, leading to a premature stop at amino acid 596; mutant Z480 has a T-to-A transversion at nt 548, resulting in premature termination at amino acid 183; and mutant G5 has a G-to-A transition at nt 4713, resulting in an E1613K missense mutation. (C) The alignment of diverse SET domains present in Drosophila demonstrates that the G5 missense mutation changes a conserved residue (E1613, marked with asterisks) thought to be required for methylase activity (Wilson et al. 2002). (D) Structural comparisons indicate significant homology between dSet1 (CG40351), Homo sapiens Set1a, and S. cerevisiae Set1.

Figure 2

dSet1 mutations result in dramatic losses of di- and trimethylated H3K4 at multiple developmental time points. H3K4 methylation levels from nuclear extracts were detected on Western blots using H3K4 methyl-specific antibodies. Extracts were normalized to total histone H3 present. The H3K4 methylation levels measured from wild-type (OreR) and dSet1 mutants (G12/γ-28) were compared across a range of developmental time points: embryonic extracts (14–20 hr after egg lay), L1 extracts (34–40 hr post egg lay), L2 larval extracts (56–64 hr post egg lay), L3 larval extracts (104–110 hr post egg lay), and pupal extracts (7–8 days post egg lay).

Figure 3

The loss of function or depletion of Drosophila COMPASS members results in reduced global H3K4 methylation. A comparison of global H3K4 methylation levels in L3 larvae containing a trans-heterozygous combination of ash2 mutant alleles (ash2¹/ash2^EY03971) or expressing double-strand RNA targeting wds, dRbbp5, dWdr82, and hcf, relative to wild-type (OreR) larvae. Levels of mRNA knockdown in RNAi experiments are represented as a percentage of wild-type levels and presented as bar graphs below.

Figure 4

dSet1 is a component of a macromolecular complex. (A) Size fractionation of adult nuclear extracts containing dSet1-2X FLAG and Ash2-3X HA on a 16–40% glycerol gradient. Fractions were recovered from top to bottom (left to right) and detected with anti-HA and anti-FLAG antibodies. The numbers listed above lanes indicate sizes (in kDa) of markers that were isolated at the respective fraction number when run on a parallel gradient. Note that the peak of dSet1-2X FLAG recovery occurs in a high-molecular-weight fraction (fraction 13, 667 kDa) and that significant levels of ash2-3X HA are also recovered in this fraction. (B) Anti-HA immunoprecipitation of Ash2-3X HA from adult nuclear extracts isolated from the genotype UAS-ash2-3X HA/+; tub-Gal4/UAS-dSet1-2X FLAG. dSet1 and Ash2 were detected with anti-FLAG and anti-HA antibodies, respectively. Note that dSet1 coprecipitates with Ash2.

Figure 5

dSet1 physically interacts with the Drosophila COMPASS members Wds and Ash2. To identify binding partners of dSet1, in vitro binding assays were performed by incubating a solution containing HA-tagged versions of dSet1 or a fragment containing the catalytic carboxy terminus of dSet1 (HA-dSet1np amino acids 1329–1641) (“input” lanes) with GST, GST-Wds, or GST-Ash2 immobilized on glutathione sepharose beads (“bind” lanes) or unbound beads (“mock” lanes), followed by washes to remove nonspecific interactors. Bound products were detected on Western blots using anti-HA and anti-GST antibodies.

Figure 6

trr, ash1, and trx mutations do not significantly alter global H3K4 methylation levels. (A) Global H3K4 levels are unaltered in trx and ash1 mutant animals. Comparison of H3K4 methylation profiles of OreR, ash1^B1/ash1^B7, and trx^B11/trx^Z11 larvae. (B) Comparison of global H3K4 methylation levels between OreR larvae and larvae expressing double-strand RNA targeting (left to right) of dSet1, trx, and trr. Levels of dSet1 and trx mRNA knockdown in RNAi experiments are represented as a percentage of wild-type levels and presented as bar graphs below. We had technical problems in quantifying the extent of knockdown in the trr RNAi line, but Mohan et al. (2011) have verified that this line shows significantly reduced levels of Trr protein. (C) H3K4 methylation defects are not observed in trr¹/trr¹ mutant haltere disc clones (marked by an absence of GFP). Clones were generated in L3 larvae of the genotype trr¹ FRT18A/Ubi-GFP FRT18A; UAS-FLP, en-Gal4/+. Note the similar amounts of H3K4 trimethylation present (red) in trr¹/trr¹ clones vs. surrounding GFP-positive tissue. (Left to right) Ubi-GFP (green); H3K4me3 staining (red); phosphotyrosine (pY) cell-surface staining (blue); and merge.