Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex

Abstract

Background: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2.

Results: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation.

Conclusion: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.

Figure 1

Alignment of the mammalian Smyd family proteins, and Smyd2 localization. (A) Schematic representation of the five mammalian Smyd proteins. The split SET domain is shown in light gray; the MYND domain is represented in black and the cysteine-rich post-SET domain is displayed in dark gray. Positions of the amino acids are indicated. (B) Expression of Smyd1, Smyd2, and Smyd3 transcripts in tissues. Top panel: Smyd3 mRNA is most highly expressed in the thymus and in skeletal muscle [8]. Middle panel: Smyd2 mRNA is most highly expressed in the heart and brain. Bottom panel: Smyd1 expression is restricted to the heart and skeletal muscle [20]. Transcripts for Smyd1, Smyd2, and Smyd3 are expressed in the embryo. (C) ClustalW alignment of the amino-terminal SET residues, the MYND domain, and the core SET residues followed by the post-SET domain present in Smyd1, Smyd2, Smyd3, Smyd4, and Smyd5. (D) Smyd2 is localized to the cytoplasm and the nucleus. Exponentially growing 293T cells were transfected with 1 μg of plasmids, encoding myc tagged Smyd2. 48 hr post-transfection, cells were fixed, washed, permeabilized and exposed to monoclonal mouse anti-myc antibody. Nuclei were counterstained with DAPI. Right panel: Smyd2 (red) localizes to both the nucleus and the cytoplasm of 293T cells. Left panel: Nuclei were counterstained with DAPI (blue). The experiments were repeated in triplicate with identical results. (E) Smyd2 mRNA is localized in the heart and hypothalamus of the brain at E13.5. Whole-mount in situ hybridization of Smyd2 transcripts in embryos at day 13.5 post coitus were prepared by exposition of sense (right panel) and antisense (middle panel) DNA probes, specific to Smyd2 to the sections. Whereas hybridization with sense probe resulted in no signal (right panel), thus, serving as control, Smyd2 mRNA is easily detected in the heart and the hypothalamus of the brain in embryos at day 13.5 post coitus (middle panel).

Figure 2

Smyd2 dimethylates Histone H3 lysine 36. (A) Smyd2 methylates histone H3 in an in vitro histone methyl transferase (HKMT) assay using mixed histones from calf thymus as substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with Histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Lanes 1 and 3 show positive HKMT activity at H3 by myc tagged Smyd3 and myc tagged Smyd2, respectively. Lanes 2 and 4 indicate that neither the Smyd3 (Y239F) nor the Smyd2 (Y240F) catalytic mutants have HKMT activity. It is concluded that the HKMT activity of Smyd3 depends on Y239 and Y240 for Smyd2. (B) Smyd2 methylates Histone H3 in an in vitro histone methyl transferase assay using recombinant octamers as substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with Histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Histone H3 was found methylated by Smyd2 using recombinant octamers as substrate in an in-vitro HKMT assay. (C) Smyd2 methylates histone H3 in an in vitro histone methyl transferase assay using recombinant histone H3 as a substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Histone H3 was found methylated by Smyd2 using recombinant octamers as substrate in an in-vitro HKMT assay (lane 1). The catalytically defective mutant Smyd2 (Y240F) failed to methylate recombinant histone H3 (lane 2). It is concluded that the HKMT activity of Smyd2 depends on Y240. (D) Smyd2 does not dimethylate histone H3 at lysine 4 using recombinant histone H3 as a substrate in an in-vitro HKMT assay. Western results, using antibodies, specifically reactive with dimethylated histone H3, lysine 4, are shown; the 17 kD band, corresponding with histone H3, is indicated. Lanes 1 and 4 indicate that immunoprecipitated and myc-tagged Smyd3, but not myc-tagged Smyd2, dimethylates histone H3 at lysine 4. Lanes 2 and 5 show that neither Smyd2 (Y240F) nor Smyd3 (Y239F) dimethylate histone H3 at lysine 4. We conclude that Smyd2 does not dimethylate histone H3 at lysine 4. (E) Smyd2 dimethylates histone H3 lysine 36 using recombinant histone H3 as a substrate in an in-vitro HKMT assay. Western results, using antibodies, specifically reactive with dimethylated histone H3, lysine 36, are shown; the 17 kD band, corresponding with histone H3, is indicated. Lanes 1 and 3 indicate that Smyd2 dimethylates recombinant histone H3 at lysine 36, independent of the myc or Gal4 tag. The catalytically inactive mutant Y240F does not dimethylate recombinant histone H3 at lysine 36 (lane 2). Smyd3, as well as the catalytically defective mutant Y239F, do not dimethylate recombinant histone H3 at lysine 36 (lanes 4 and 5). We conclude that Smyd2 dimethylates recombinant histone H3 at lysine 36, whereas Smyd3 does not display this activity.

Figure 3

Smyd2 associates with the Sin3 repression complex and is involved in transcriptional repression. (A) Expression of GAL4-Smyd2 fusion protein in 293T cells. Exponentially grown 293T cells were transfected with the constructs indicated and, 48 hours post transfection, whole cell lysate was prepared using RIPA buffer and subjected to western analysis using antibodies directed against the GAL4 tag. A reactive band was detected at the appropriate molecular weight (approximately 66 kD). Extracts from cells, transfected with the GAL4-DBD construct [17], served as negative control. (B) Smyd2 represses transcription of a luciferase reporter. Top panel: Schematic illustration of the reporter construct used. Bottom panel: 10T1/2 cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 μg) together with GAL4-DBD or GAL4-Smyd2 (2 μg each). Percent activity of the luciferase was determined in relation to GAL4-DBD. Smyd2 significantly represses the transcription of a luciferase reporter in 10T1/2 cells. (C) Smyd2 associates with HDAC1. Exponentially grown 293T cells were transiently co-transfected with GAL4-DBD or GAL4-Smyd2, together with Flag tagged HDAC1 (HDAC1-F). Whole cell RIPA extracts were immunoprecipitated using an anti-GAL4 antibody and immunoblots were probed with an anti-FLAG antibody. As shown here, Smyd2 associates with HDAC1. RIPA whole cell extracts from GAL4-DBD transfected cells [17] served as negative control. Equal protein amounts in the immunopreciptation assays was demonstrated by analysis of 5% input using anti Flag antibodies. (D) Smyd2 interacts with the Sin3A but not the NuRD complex. Exponentially grown 293T cells were transfected with the constructs indicated and, 48 hours post transfection, whole RIPA lysate was prepared. Antibodies directed against GAL4 were used for immunoprecipitation, followed by western analysis using the antibodies indicated. Smyd2 associates with HDAC1 and Sin3A but not with the components of the NuRD complex, MBD3 or MTA2.

Figure 4

Smyd2 suppresses NIH3T3 cell proliferation. Exponentially grown NIH3T3 cells were transfected with plasmids encoding myc-tagged Smyd2 or myc-tagged Smyd3. Cells, transfected with the empty expression construct (Mock), served as control. All cells were monitored by cell counting using trypan blue exclusion. The inserts show the level of expression of Smyd2-myc and Smyd3-myc at 0 and 144 hours post transfection, demonstrating similar levels of ectopically introduced proteins in the NIH3T3 cell lines used. Whereas ectopically introduced Smyd3 enhanced the proliferation, Smyd2 displayed a negative effect on the growth rate of NIH3T3 cells.