P.16.7 Length dependent telomere looping affects long-distant gene expression (5Mb) in FSHD

Abstract

The genetic locus of Facioscapulohumeral Muscular Dystrophy (FSHD) is only ∼25–50 kb from the telomere of Chr.4q, and shows an age-related onset of symptoms. This suggested that Telomere Position Effects (TPE) might regulate its poorly understood pathogenetic mechanism. FSHD patients show a reduction in the number of 3.3 kb D4Z4 repeats on at least one copy of chromosome 4. We first showed that continuous spreading of telomeric heterochromatin (classic TPE) could extend at least 100 kb into the subtelomeric region and increased Dux4-fl expression >10-fold when telomeres were short. We then examined whether telomere looping existed/influenced gene expression over much larger distances. We adapted the Hi–C technique to study the 4q region in an unbiased fashion. We established clonal myoblast cell lines containing a floxable hTERT that we removed at different times to initiate telomere shortening, producing isogenic subclones with different telomere lengths. We identified multiple novel long-range interactions at 4q associated with telomere shortening. We validated one interaction using 3C and 3D co-FISH experiments. A 4.8 Mb DNA loop present in FSHD myoblasts with long telomeres (20 Kb) and in cells obtained from unaffected age matched siblings (regardless of telomere length) was lost on the FSHD contracted allele when telomeres became shorter (9 Kb,long before telomeres became short enough to induce replicative senescence). Intriguingly, interactions involved several putative FSHD genes and a gene supposedly expressed only in cardiac muscle. Droplet-digital PCR (ddPCR) demonstrated changes in the abundance of different exon-pairs for a variety of candidate genes affecting FSHD in cells with short vs long telomeres, indicating telomere looping was at least altering splicing patterns if not overall transcription. These results suggest a role for telomere looping in FSHD pathology that could explain much of the age-related onset and variability in this disease.