Identification of SMCHD1 domains for nuclear localization, homo-dimerization, and protein cleavage

Abstract

Background: SMCHD1 is a disease modifier and a causative gene for facioscapulohumeral muscular dystrophy (FSHD) type 1 and type 2, respectively. A large variety of different mutations in SMCHD1 have been identified as causing FSHD2. In many cases, it is unclear how these mutations disrupt the normal function of SMCHD1.

Methods: We made and analyzed lenti-viral vectors that express Flag-tagged full-length or different mutant SMCHD1 proteins to better understand the functional domains of SMCHD1 in muscle cells.

Results: We identified regions necessary for nuclear localization, dimerization, and cleavage sites. Moreover, we confirmed that some mutants increased DUX4 expression in FSHD1 myoblasts.

Conclusions: These findings provide an additional basis for understanding the molecular consequences of SMCHD1 mutations.

Keywords: DUX4; Facioscapulohumeral muscular dystrophy; Homo-dimerization; Nuclear localization; Protein cleavage; SMCHD1.

Fig. 1

Schematic of full-length and different mutant SMCHD1 constructs. The gene and protein SMCHD1 consists of 48 exons and 2005 amino acids, respectively, and the ATPase domain in the amino-terminus and the hinge domain in the carboxy-terminus are indicated in red and blue, respectively. A bar indicates recognition regions for anti-SMCHD1 antibody (HPA039441 and ab179456). Predicted molecular weight is shown in the right side. 3 × HA tag (green) and 3 × Flag tag (yellow)

Fig. 2

Identification of the nuclear localization signal. a Immunofluorescence for SMCHD1 in control myoblasts. b–d Immunofluorescence for Flag in control myoblasts transduced with Flag-tagged SMCHD1 lentivirus. DAPI was used for staining the nucleus. Scale bar is 20 μm. The regions necessary for nuclear localization were confirmed by repeat experiments

Fig. 3

Single spot of SMCHD1 in female myotube. a Immunofluorescence for SMCHD1 and myosin heavy chain (MF20 antibody) in control female myotubes. b Immunofluorescence for SMCHD1 and H3K27me3 in control female myotubes. c Immunofluorescence for Flag in control female myotubes transduced with Flag-tagged SMCHD1 lentivirus. DAPI was used for staining the nucleus. Scale bar is 20 μm. Single spot of full length was confirmed by repeat experiments

Fig. 4

Identification of regions necessary for SMCHD1 homo-dimerization. a, b IP of exogenous SMCHD1 in control myoblasts transduced with Flag-tagged SMCHD1 lentivirus followed by Western blot with an anti-SMCHD1 antibody (ab179456) detecting the carboxy-terminal region of SMCHD1. For confirming the expression of Exon1-36-Flag, anti-Flag antibody was used after stripping. α-Tubulin was used as a loading control. Red and blue arrows identify endogenous and exogenous SMCHD1, respectively. Blue double arrows identify smaller fragments of exogenous SMCHD1. IgG heavy chain (**) and light chain (*). IP (immunoprecipitation)

Fig. 5

DUX4 expression in FSHD1 myoblasts with mutant SMCHD1. RT-qPCR for DUX4, ZSCAN4, MBD3L2, and SMCHD1 in FSHD1 myoblasts transduced with different mutant SMCHD1 constructs. RPL27 was used as an internal control. (n = 5 in each group) Dunnett’ s test (*P < 0.01). These are representative data from three experiments

Fig. 6

Identification of the cleavage sites. a, b, and d–f Western blot for SMCHD1 (HPA039441 and ab179456) and Flag in control myoblasts transduced with Flag-tagged SMCHD1. (c) Western blot for HA and Flag in control myoblasts transduced with HA-Exon1-14.47M-48-Flag. α-Tubulin was used as a loading control. Red and blue arrows indicate endogenous and exogenous SMCHD1, respectively. Red and blue double arrows indicate smaller fragments of endogenous and exogenous SMCHD1, respectively. 10A.11D (Exon1-10A.11D-14.47M-48). 11E.12D (Exon1-11E.12D-14.47M-48). The smaller band from full length was confirmed by repeat experiments