SMYD4 interacts with HDAC1.
(A) Immunofluorescence staining and representative confocal images of the sub-cellular localization of SMYD4 in HL-1 cells. SMYD4 was found in both the nucleus and cytoplasm. The experimental groups without primary anti-SMYD4 antibody incubation served as negative controls; (B) Co-IP/western blotting analysis to confirm the interaction between SMYD4 and HDAC1. SMYD4flag was overexpressed in HL-1 cells. The cell extracts were immunoprecipitated with an anti-FLAG affinity gel, followed by western blotting analysis; (C) The interaction between SMYD4 and HDAC1 was further confirmed by Co-IP/western blotting analysis in HEK293T cells; (D-E) Using a Co-IP/western blotting assay to map the domains in SMYD4 that are responsible for the interaction with HDAC1. The MYND domain of SMYD4 was found to be critical for this interaction; (F) Aberrant histone modifications in MZsmyd4L544Efs*1 embryos at 48 hpf. Specifically, H3K4me2 and H3K4me3 were significantly reduced, and H3K4me was increased, while H3K9me3 and H3K27me3 were not affected, suggesting that SMYD4 is a H3K4-specific methyltransferase. H3K4ac, H3K9ac, H3K14ac, and H3K27ac were significantly abolished in MZsmyd4L544Efs*1 mutants, suggesting the increased activity of HDAC1 in MZsmyd4L544Efs*1 mutants and confirming SMYD4 as an important negative regulator of HDAC1 function; (G) The semi-quantitative analysis of the histone modifications changes in MZsmyd4L544Efs*1 mutants. (p<0.05 *, p<0.01 **).