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 . 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 . 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).