Testing the position-effect variegation hypothesis for facioscapulohumeral muscular dystrophy by analysis of histone modification and gene expression in subtelomeric 4q

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a unique dominant disorder involving shortening of an array of tandem 3.3 kb repeats. This copy-number polymorphic repeat, D4Z4, is present in arrays at both 4q35 and 10q26, but only 4q35 arrays with one to 10 copies of the repeat are linked to FSHD. The most popular model for how the 4q35 array-shortening causes FSHD is that it results in a loss of postulated D4Z4 heterochromatinization, which spreads proximally, leading to overexpression of FSHD genes in cis. This would be similar to a loss of position-effect variegation (PEV) in Drosophila. To test for the putative heterochromatinization, we quantitated chromatin immunoprecipitation with an antibody for acetylated histone H4 that discriminates between constitutive heterochromatin and unexpressed euchromatin. Contrary to the above model, H4 acetylation levels of a non-repeated region adjacent to the 4q35 and 10q26 D4Z4 arrays in normal and FSHD lymphoid cells were like those in unexpressed euchromatin and not constitutive heterochromatin. Also, these control and FSHD cells displayed similar H4 hyperacetylation (like that of expressed genes) at the 5' regions of 4q35 candidate genes FRG1 and ANT1. Contrary to the loss-of-PEV model and a recent report, there was no position-dependent increase in transcript levels from these genes in FSHD skeletal muscle samples compared with controls. Our results favor a new model for the molecular genetic etiology of FSHD, such as, differential long-distance cis looping that depends upon the presence of a 4q35 D4Z4 array with less than a threshold number of copies of the 3.3 kb repeat.

Common epigenetic changes of D4Z4 in contraction-dependent and contraction-independent FSHD

“…This argues against a cis spreading effect from D4Z4 in proximal direction. Also analysis of histone modifications in 4qter argues against this disease model for FSHD [22] . We propose that the recently identified CTCF binding site at the proximal end of the D4Z4 repeat (G. Filippova, personal communication) may prevent spreading of hypomethylation in a proximal direction.…”

“…The absence of FRG2 on the disease alleles in some FSHD patients with a proximal deletion makes this gene a less attractive candidate gene [39] . However, transcriptional deregulation of these candidate genes in patients with FSHD is still under debate, as several studies showed contradictory results [22,[40][41][42][43] . Based on our data showing that hypomethylation is restricted to the D4Z4 repeat and not spreading in a proximal direction in addition to previous data showing no change in the chromatin structure of proximal sequences in FSHD [22] , we do not support a cisspreading mechanism emanating from D4Z4 in FSHD.…”

“…The D4Z4 repeat array is frequently considered heterochromatic because of the presence of the low-copyrepeats hhspm3 and LSau, repetitive sequences that are mainly found in heterochromatic regions of the human genome [21] . However, several studies have indicated that D4Z4 has both euchromatic and heterochromatic features [22,23] . Interestingly, DNA methylation of the proximal D4Z4 repeat unit on the disease allele was significantly reduced in patients with FSHD1, both in DNA isolated from peripheral blood lymphocytes (PBLs) and from muscle [24] .…”

D4Z4as a prototype of CTCF and lamins-dependent insulator in human cells

“…[42][43][44][45] This proximal anchoring may provide a relatively high mobility of the 4q end, which has a large diffusion volume, change by the length of D4Z4 array (Fig. 3).…”

No effect of folic acid and methionine supplementation on D4Z4 methylation in patients with facioscapulohumeral muscular dystrophy

“…Changes in levels and patterns of DNA methylation also have an important role in oncogenesis [3] . In healthy individuals, the chromatin structure of the D4Z4 region on chromosome 4q35 is highly methylated, and resembles that of heterochromatin or transcriptional inactive euchromatin [4][5][6] . A currently favored hypothesis is that the marked hypomethylation of the D4Z4 region in FSHD might cause a change in the chromatin structure, and consequently a transcriptional deregulation of one or more genes in the vicinity, or at a distance of the D4Z4 repeat array [1,6] .…”