Genetic and Epigenetic Characteristics of FSHD-Associated 4q and 10q D4Z4 that are Distinct from Non-4q/10q D4Z4 Homologs

ABSTRACT Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri‐methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non‐4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD. Linking epigenetic alteration and DUX4 expression in FSHD. (A) SMCHD1 is a component of the H3K9me3 heterochromatin at D4Z4 that represses the DUX4 gene. The loss of H3K9me3,an epigenetic change directly linked to FSHD,upregulatesDUX4. (B) In contrast to D4Z4 on chromosomes 4q and 10q, D4Z4 homologs on seven other chromosomes are highly heterogeneous with frequent interruptions of the DUX4 ORF and do not undergo the epigenetic alteration in FSHD. This underscoresthe unique characteristics of 4q/10q D4Z4 among the related repeats.