DUX4 Binding to Retroelements Creates Promoters That Are Active in FSHD Muscle and Testis

Abstract

The human double-homeodomain retrogene DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression of DUX4 in somatic tissues and result in mis-expression of this transcription factor in skeletal muscle. DUX4 binds sites in the human genome that contain a double-homeobox sequence motif, including sites in unique regions of the genome as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on myoblasts transduced with DUX4 we show that DUX4 binds and activates transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for genes, long non-coding RNAs, and antisense transcripts. Many of these novel transcripts are expressed in FSHD muscle cells but not control cells, and thus might contribute to FSHD pathology. For example, HEY1, a repressor of myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs, including those in repetitive elements, show evolutionary conservation and some repeat-initiated transcripts are expressed in healthy testis, the normal expression site of DUX4, but more rarely in other somatic tissues. Testis expression patterns are known to have evolved rapidly in mammals, but the mechanisms behind this rapid change have not yet been identified: our results suggest that mobilization of MaLR and ERV elements during mammalian evolution altered germline gene expression patterns through transcriptional activation by DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in mammalian germline evolution and in FSHD muscular dystrophy.

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle

“…These cultures were described previously (26). As previously described, primary myoblast cell lines were received from the University of Rochester biorepository (http://www.urmc.…”

Facioscapulohumeral Muscular Dystrophy As a Model for Epigenetic Regulation and Disease

“…Although DUX4-S regulates expression of a much smaller set of genes (55), both isoforms contain the same double homeobox DNA binding domain and are thought to function as transcription factors (43,55,175). However, only expression of the DUX4-FL isoform is linked to FSHD (78,96,143) and DUX4-FL-specific target genes, which include genes expressed in the germline and in early development, immune mediators (e.g., b-defensin 3), and retroelements (e.g., MaLRs), are misregulated in FSHD (55,175). Therefore, FSHD involves both an increase in DUX4 gene transcription and a switch in DUX4 alternative splicing.…”

“…Low levels of DUX4-FL are highly cytotoxic when expressed in somatic cells or during vertebrate development (20,89,111,164,171), and DUX4-FL expression in myogenic cells disrupts differentiation and causes the atrophic myotube phenotype found in FSHD myotubes (19,162). Although the mechanisms are still unclear, it is thought that aberrant expression of DUX4 targets (immune mediators, germline genes, and the products of DUX4-activated retroelements) leads to muscle pathology (55,175). Two myogenic enhancers proximal to D4Z4 were recently identified and shown to regulate DUX4 (70), providing a potential explanation for the relatively muscle-specific pathology seen in FSHD.…”

DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis

“…Although DUX4 binding motifs have been identified (Dixit et al, 2007;Ferri et al, 2015;Geng et al, 2012;Young et al, 2013;Choi et al, 2016), and ChIP-Seq performed (Geng et al, 2012;Choi et al, 2016), a set of target genes that explains both anti-myogenic and apoptotic phenotypes induced by DUX4 has not been comprehensibly defined.…”