Smyd1 Orchestrates Early Heart Development Through Positive and Negative Gene Regulation

Abstract

SET and MYND domain-containing protein 1 (Smyd1) is a striated muscle-specific histone methyltransferase. Our previous work demonstrated that deletion of Smyd1 in either cardiomyocytes or the outflow tract (OFT) resulted in embryonic lethality at E9.5, with cardiac structural defects such as truncation of the OFT and right ventricle and impaired expansion and proliferation of the second heart field (SHF). The cardiac phenotype was accompanied by the downregulation of ISL LIM Homeobox 1 (Isl1) and upregulation of atrial natriuretic factor (ANF). However, the mechanisms of Smyd1 regulating Isl1 and ANF during embryonic heart development remain to be elucidated. Here, we employed various biochemical and molecular biological approaches including chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR), pGL3 fluorescence reporter system, and co-immunoprecipitation (CoIP) and found that Smyd1 interacted with absent small homeotic-2-like protein (ASH2L) and activated the promoter of Isl1 by trimethylating H3K4. We also found that Smyd1 associated with HDAC to repress ANF expression using trichostatin A (TSA), a deacetylase inhibitor. In conclusion, Smyd1 participates in early heart development by upregulating the expression of Isl1 and downregulating the expression of ANF.

Keywords: ANF; ASH2L; ISL-1; Smyd1; heart.

FIGURE 1

The expression of Smyd1 in mouse embryos. (A–C) Whole-mount in situ hybridization reveals the specific expression of Smyd1 in hearts at E8.5, E9.0, and E9.5. (D) In situ analysis reveals the expression of mouse Smyd1 in transverse sections at E10.0. HT, heart tube; OFT, outflow tract; LV, left ventricle; V, ventricle; RV, right ventricle.

FIGURE 2

Smyd1 binds to the promoter of Isl1. (A) The locations of the primer sets (P1-TSS) used for Smyd1 ChIP-qPCR are indicated. (B) The sequence of the mouse Isl1 gene containing the SB4-binding site is shown and highly conserved across mouse, rat, and human. (C) ChIP-qPCR revealed that Smyd1 bound to the P4 region avidly. (D) EMSA was performed using nuclear extracts from CV1 cells overexpressing Smyd1. The radiolabeled SB4 site shown was used as a probe. WT and MUT competitors were used at amounts from 5 × to 200 × fold molars. (E) Sequences that were mutated on the Isl1 promoter were indicated by arrows. Smyd1 expression vectors were cotransfected with pLG3-Isl1-WT or mutant reporter (Isl1 SB4MUT or Isl1SB3MUT) into C2C12 cells. Data are shown as mean ± SD for three independent experiments. *p < 0.01 (Student’s t test).

FIGURE 3

Smyd1 was responsible for H3K4me3 in the P4 region of the Isl1 promoter. (A,B) ChIP-qPCR for the H3K4me3 and H3K27me3 level on the different regions of the Isl1 promoter. (C) Sequences that were mutated on mouse Smyd1 cDNA to generate a mutation in the HMT catalytic domain of Smyd1. Amino acid Y234 was mutated to F (Y234F). Smyd1 WT or Y234F expression vectors were cotransfected with the pLG3-Isl1 reporter. Data are shown as mean ± SD for three independent experiments. *p < 0.01, **p < 0.01 (Student’s t test).

FIGURE 4

ASH2L interacts with Smyd1. (A) Lacz staining shows the expression pattern of ASH2L at E9.5. (B) Superdex 200 gel filtration revealed formation of a complex containing ASH2L and Smyd1. (C) Western blot detected ASH2L and Smyd1. (D) V5-tagged ASH2L and Myc-tagged Smyd1 were immunoprecipitated with an anti-V5 antibody. Normal IgG was used as a control. (E) Smyd1 and ASH2L locations in murine heart sections were determined by immunofluorescence. They were co-expressed in the nuclei and cytoplasm of cardiomyocytes with local overlapping regions (arrow). (F) Smyd1 WT or Y234F was cotransfected with ASH2L and pLG3-Isl1-WT luciferase reporter into C2C12 cells. The scale is 10 μm. Data are shown as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01 (Student’s t test).

FIGURE 5

Smyd1 associates with HDAC to repress ANF expression. (A) The locations of the primer sets used for Smyd1 ChIP-qPCR are indicated. (B) The ChIP-qPCR results revealed that Smyd1 bound to the P2 region avidly. (C) Smyd1 expression vectors were cotransfected with pLG3-ANF-WT or mutant reporter (ANF MUT) into CV1 cells. (D) pLG3-ANF vectors were cotransfected with a different dosage of HDAC1 or Smyd1 into CV1 cells. (E) A different dose of TSA was added to pLG3-ANF/HDAC1 or pLG3-ANF/Smyd1-cotransfected cells. Data are shown as mean ± SD for three independent experiments. *p < 0.05 (Student’s t test).