Facioscapulohumeral muscular dystrophy (FSHD) is a consequence of the mis-expression of the DUX4 protein in skeletal muscle, causing muscle wasting, or dystrophy. Intriguingly, there are two forms of FSHD. The more common form, FSHD1, is autosomal dominant, and is caused by a shortened D4Z4 macrosatellite repeat array where there are fewer than ten copies of the DUX4 gene, resulting in chromatin relaxation. Patients with FSHD2, the less common form, however, have a normal number of DUX4 copies within the macrosatellite array, yet there is still significant relaxation of the D4Z4 chromatin to result in variegated expression of DUX4 resulting in wasting of muscle cells. This illustrates that at the disease mechanism level, there is a fundamental difference between FSHD1 and FSHD2; however DUX4 still gets expressed in both forms of disease, resulting in identical clinical manifestations. A deeper understanding of the genetic underpinnings of the disease will give the research community a better grasp of FSHD. The Tapscott lab (Human Biology Division) and others have previously identified FSHD2 to be caused by mutations in SMCHD1, a chromatin factor on chromosome 18 that regulates repression and is required for silencing certain repetitive regions, including the D4Z4 array. SMCHD1 segregates independently from the DUX4 allele located on chromosome 4, resulting in a digenic inheritance in patients. FSHD1 patients who happen to have mutations in SMCHD1 have more severe disease, suggesting that the SMCHD1 gene has the ability to affect the severity of disease. Since the initial observation, several FSHD2-causing mutations within SMCHD1 have been reported. Nevertheless, to better understand the functional consequences of these mutations, it is imperative to gain a deeper knowledge of the functional regions of the SMCHD1 protein. Dr. Yosuke Hiramuki, a postdoctoral fellow in the Tapscott lab, systematically studied the role of SMCHD1 as a chromatin binding protein, and published this work in a recent issue of Skeletal Muscle.
The authors focused their study on identifying the regions necessary for nuclear localization and homo-dimerization, key regions for chromatin binding. Dr. Hiramuki constructed lentiviral vectors expressing full length or a variety of deletion SMCHD1 mutants, and expressed these constructs in muscle cells. Immunofluorescence was used to identify the nuclear localization signal. Previous reports identified the hinge domain to be important for SMCHD1 homo-dimerization. The authors similarly used immunoprecipitation (IP) to determine whether different SMCHD1 mutants could dimerize with endogenous SMCHD1, and found that the hinge domain can mediate dimerization between endogenous and mutant SMCHD1 proteins. Interestingly, the expression of these mutant SMCHD1 proteins did not alter the abundance of the endogenous SMCHD1, suggesting mutations in SMCHD1 may not affect the expression of the protein, but instead it may alter its function by forming inactive heterodimers.
In the experiments, the authors were surprised to observe smaller proteins in addition to the predicted mutant SMCHD1 proteins. The production of these smaller proteins may be attributed to the presence of proteolytic cleavage sites. Dr. Hiramuki further tested a series of mutant SMCHD1 vectors to pinpoint the sites of cleavage, and speculated that these specific cleavage sites may be found in the endogenous SMCHD1.
This study seeks to identify the domains that confer functional interactions in the SMCHD1 protein, such as the nuclear localization signal and homo-dimerization domains. Understanding these domains will be important for unraveling the molecular mechanisms of mutant SMCHD1 regulation of DUX4 expression in FSHD2. Dr. Hiramuki describes what lies beyond this study: “As a next step, we need to develop our findings into understanding how SMCHD1 mutations are associated with the onset of FSHD. Taking into account that many types of SMCHD1 mutations have been reported in FSHD without an apparent hot spot, we are now seeking to identify the difference between wild-type and FSHD-causing SMCHD1 mutations, not only to understand the pathological mechanisms but also to develop treatments for FSHD patients.”
Hiramuki Y and Tapscott SJ. 2018. Identification of SMCHD1 domains for nuclear localization, homo-dimerization and protein cleavage. Skeletal Muscle Aug 2; 8(1):24
Funding was provided by the FSH Society and FSHD Canada Foundation.