J Cell Biol-2010-Cabianca-1049-60(2)

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The Rockefeller University Press $30.00J. Cell Biol. Vol. 191 No. 6 10491060

www.jcb.org/cgi/doi/10.1083/jcb.201007028 JCB 104

JCB: Review

Correspondence to Davide Gabellini: [email protected]

Abbreviations used in this paper: 3C, chromosome conormation capture; CNV,copy number variation; DRC, D4Z4 repressor complex; FSHD, acioscapulo-humeral muscular dystrophy; H3K9me3, histone H3 lysine 9 tri-methylation;H3K27me3, histone H3 lysine 27 tri-methylation; MAR, matrix attachment re-gion; SNP, single-nucleotide polymorphism.

Copy number variations are an important

source o human genetic diversity

Genetic association studies generally evaluate single-nucleotide

polymorphisms (SNPs), which are single nucleotides at specic

genomic locations that vary between individuals o the same

species. Recent results indicate that the human genome contains

another requent type o polymorphism: copy number varia-

tions (CNVs; Conrad et al., 2010). A CNV is a segment o DNA

that can be ound in various copy numbers in the genomes o

dierent individuals (Fig. 1). CNVs range in size rom a ew

hundred nucleotides to several megabases. Compared with SNPs,

CNVs aect a more signicant raction o the genome and

arise more requently. Hence, CNVs signicantly contribute to

human evolution, genetic diversity, and an increasing number o

phenotypic traits (Stankiewicz and Lupski, 2010).

Depending on the genomic context, CNVs can have vary-

ing eects (Fig. 1). For example, recent data indicate that CNVs

directly alter the structure o 12.5% o protein-coding genes

(Conrad et al., 2010), and there is increasing evidence to suggest

that CNVs play an important role in a number o Mendelian dis-

eases and common complex disorders (Stankiewicz and Lupski,2010). For example, Charcot-Marie-Tooth type 1A disease is

caused by duplications in the PMP22 gene, which encodes an inte-

gral membrane protein that is a major component o compact my-

elin in the peripheral nervous system (Chance et al., 1994). Also,

susceptibility to acquired immune deciency syndrome is aected

by segmental duplications encompassing the CCL3L1 gene,

which encodes the CCR5 chemokine and ligand or the human

immunodeciency virus coreceptor (Gonzalez et al., 2005).

CNVs can also aect gene dosage and expression (Stranger

et al., 2007). Recent data indicate that nonB-DNA orming se-

quences, which are usually enriched in promoter regions, are

also enriched in CNV breakpoints. Thus, the same eatures that

are involved in transcriptional regulation may also be involvedin the ormation o CNVs. As a consequence, CNVs might shape

the evolution o gene regulation (Conrad et al., 2010).

More than hal o the human genome is comprised o repeti-

tive sequences (Neguembor and Gabellini, 2010). Because repeti-

tive sequences can act as substrates or homologous recombination,

their presence acilitates the instability o our genome (Gu et al.,

2008). As a result, repetitive sequences account or a signicant

amount o human CNVs (Warburton et al., 2008).

In this review we will ocus on an important human ge-

netic disease, acioscapulohumeral muscular dystrophy (FSHD),

which is caused by the presence o CNVs o a repetitive sequence

that regulates gene expression.

Clinical eatures o FSHD

FSHD (MIM #158900) is characterized by the progressive

weakness and atrophy o a specic subset o skeletal muscles.

In humans, copy number variations (CNVs) are a com-mon source o phenotypic diversity and disease suscepti-bility. Facioscapulohumeral muscular dystrophy (FSHD) isan important genetic disease caused by CNVs. It is an

autosomal-dominant myopathy caused by a reduction inthe copy number o the D4Z4 macrosatellite repeat lo-cated at chromosome 4q35. Interestingly, the reduction oD4Z4 copy number is not sufcient by itsel to cause FSHD.A number o epigenetic events appear to aect the sever-ity o the disease, its rate o progression, and the distribu-tion o muscle weakness. Indeed, recent fndings suggestthat virtually all levels o epigenetic regulation, rom DNAmethylation to higher order chromosomal architecture,are altered at the disease locus, causing the de-regulationo 4q35 gene expression and ultimately FSHD.

The cell biology o disease

FSHD: copy number variations on the theme omuscular dystrophy

Daphne Selvaggia Cabianca1,2 and Davide Gabellini2,3

1International PhD Program in Cellular and Molecular Biology, Vita-Salute San Raaele University, 20132 Milan, Italy2Division o Regenerative Medicine, San Raaele Scientifc Institute, DIBIT 1, 2A3-49, 20132 Milan, Italy3Dulbecco Telethon Institute, 20132 Milan, Italy

2010 Cabianca and Gabellini This article is distributed under the terms o an AttributionNoncommercialShare AlikeNo Mirror Sites license or the frst six months ater the pub-lication date (see http://www.rupress.org/terms). Ater six months it is available under aCreative Commons License (AttributionNoncommercialShare Alike 3.0 Unported license,as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Published December 13, 2010


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JCB VOLUME 191 NUMBER 6 20101050

onset can vary rom inancy to age 50 (van der Maarel et al.,

2007). Early-onset cases are generally associated with more se-

vere phenotypes (Miura et al., 1998; Klinge et al., 2006). Inter-estingly, the FSHD phenotype is gender dependent. Typically,

males are more severely aected, whereas emales can develop

a milder or asymptomatic orm o the disease (Padberg, 1982;

Zatz et al., 1998; Ricci et al., 1999; Tonini et al., 2004).

Muscle impairment in FSHD is oten asymmetric: mus-

cles on one side o the body appear much more compromised

than on the other side (Kilmer et al., 1995). Various hypotheses

have been proposed to explain this phenomenon, including over-

work weakness and handedness, but the mechanism underlying

this asymmetric phenotype in FSHD remains unknown (Pandya

et al., 2008).

The rate o FSHD progression and the distribution o

muscle weakness are highly variable, even between close amily

relatives. Indeed, these eatures were noted in the rst study

conducted on the disease in the late 1800s (Landoyzy and

Dejerine, 1886). A number o monozygotic twins discordant or

the penetrance o FSHD have been described, pointing to a

strong epigenetic component in the disease (Tawil et al., 1993;

Griggs et al., 1995; Hsu et al., 1997; Tupler et al., 1998).

Although the genetic deect underlying FSHD has been

identied, the molecular mechanism causing the disease re-

mains unclear. Recent results suggest that complex genetic

events contribute to FSHD, as discussed below.

As the name implies, FSHD mostly aects the muscles o the

ace, scapula, and upper arms (Tawil et al., 1998). The peculiar

involvement o specic muscles is such a striking eature oFSHD that it is oten used in the clinic to distinguish FSHD

rom the other orms o muscular dystrophy (Padberg et al.,

1991). FSHD onset generally involves the wasting o acial

muscles, such as orbicularis oris and orbicularis oculi, whereas

others, like the pharyngeal and lingual muscles, are unaected.

As the disease progresses, limb girdle muscles, such as scapula

xator and trapezius, are also aected. Abdominal muscle weak-

ness is another eature o FSHD, causing a characteristic lordotic

posture (an abnormal curvature o the lumbar spine) associated

with a protuberant abdomen. In the most severe cases, the

muscular degeneration can extend to the pelvic girdle and oot

dorsifexor muscles, thereby aecting the ability o the patient

to walk. Approximately 20% o FSHD patients become wheel-

chair bound (Pandya et al., 2008).

FSHD is associated with retinal vasculopathy, a blood vessel

disorder o the retina, in 60% o cases (Tawil and Van Der Maarel,

2006) and sensorineural hearing loss in 75% o aected indi-

viduals (Trevisan et al., 2008). Mental retardation, epilepsy, and

cardiac involvement are also present in FSHD patients more re-

quently than in healthy people (Faustmann et al., 1996; Funakoshi

et al., 1998; Trevisan et al., 2006, 2008; Saito et al., 2007).

Most FSHD patients report their rst symptoms during

the second or third decade o their lie; however, the age o

Figure 1. CNVs and their possible effects on gene expression. CNVs can generate dierent outcomes based on the nature o the aected element. I aCNV encompasses an entire gene(s) locus, the dosage o the gene may be altered, leading to gene amplifcation or deletion. Alternatively, the aectedregion can encompass only part o a gene. In this case, i the CNV includes an exon, it can result in the production o an aberrant protein isoorm. Whena CNV localizes to a nonprotein coding region o the gene it may generate imbalances at the level o splicing or noncoding RNA (ncRNA) production.In addition, extragenic CNVs can give rise to altered gene expression when aecting cis regulatory regions.



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10Copy number variations and FSHD Cabianca and Gabellini

A detailed genomic characterization o the 4q35 region ledto the identication o dierent haplotypes (Fig. 3; van Geel

et al., 2002; Lemmers et al., 2007, 2010a). A simple sequence

length polymorphism is localized 3.5 kb proximal to D4Z4.

D4F104S1 (p13E-11), a region located immediately proximal to

D4Z4, contains 15 SNPs. The most proximal unit o the D4Z4

repeat array contains several SNPs. Finally, a large region o

sequence variation (alleles A, B, or C) has been detected distal

to D4Z4 (Fig. 3). Considering these various eatures, 4q alleles

were subdivided in 18 haplotype variants (Lemmers et al., 2010a).

Importantly, D4Z4 deletions are pathogenic only in a ew o

these haplotype backgrounds (4qA161, 4qA159, and 4qA168;

Lemmers et al., 2007, 2010b). D4Z4 deletions in the presence

o these haplotypes are not sucient to cause FSHD because

4qA161 asymptomatic carriers have been described (Arashiro

et al., 2009), suggesting that these haplotypes represent only a

permissive condition or FSHD rather than being the causative

event. Importantly, it was observed that FSHD2 patients carry at

least one 4qA161 allele (de Gree et al., 2009), urther supporting

the role o this permissive haplotype in the disease.

There are sequences homologous to D4Z4 on several

human chromosomes (Lyle et al., 1995). On chromosome 10q26

there is a repeat array that shares 98% identity with the D4Z4

repeat array at 4q35 (Cacurri et al., 1998). Additionally, high

homology extends to 45 kb proximal o D4Z4 and 1525 kb

distal (van Geel et al., 2002). The 10q and 4q D4Z4 repeats areequally polymorphic, and some individuals have nonstandard,

hybrid alleles containing 4q-derived repeats on chromosome 10

and 10q repeats on chromosome 4 (van Deutekom et al., 1996a;

van Overveld et al., 2000; Lemmers et al., 2010a). Several stud-

ies have reported that the D4Z4 repeats (even i 4q derived) on

10q are not pathogenic, suggesting that 4q-specic sequences

proximal to D4Z4 are required or FSHD (Bakker et al., 1995;

Deidda et al., 1996; van Deutekom et al., 1996a; Lemmers

et al., 1998; van Overveld et al., 2000). By contrast, an excep-

tion to this rule was recently described (Lemmers et al., 2010b).

In this unusual case, only the distal end o the D4Z4 repeat array

FSHD genetics

FSHD is the third most common myopathy, with an incidence

in the general population o 1:15,000 (Flanigan et al., 2001).

The disease is transmitted as an autosomal-dominant character,

although it presents very complex genetics. Up to 30% o cases

are due to de novo mutations (Zatz et al., 1995). Approximately

hal o the de novo cases result rom a post-zygotic mutation

that leads to mosaicism (Griggs et al., 1993; Weienbach et al.,

1993; Upadhyaya et al., 1995; Bakker et al., 1996; van der

Maarel et al., 2000). In more than 95% o cases, the disease

maps to the subtelomeric region o chromosome 4 long arm at

4q35.2 (FSHD1; Wijmenga et al., 1990, 1991). A small percent-

age o FSHD cases are not genetically linked to chromosome 4

(FSHD2; Wijmenga et al., 1991; Gilbert et al., 1992; Bakker

et al., 1995), although no other putative genetic locus has

been identied.

In 4q-associated FSHD cases, the disease is caused by a

molecular rearrangement that causes copy number variations o

a 3.3-kb tandem repeated macrosatellite called D4Z4 (Wijmenga

et al., 1992; van Deutekom et al., 1993). D4Z4 is extremely

polymorphic in the general population (Hewitt et al., 1994;

Winokur et al., 1994), ranging rom 11 to 150 copies. FSHD

patients carry only 1 to 10 units (Fig. 2; Wijmenga et al., 1992;

van Deutekom et al., 1993).

Although FSHD is highly variable, there is a general cor-

relation between the number o residual D4Z4 repeats, the ageo onset, and the severity o the disease (Goto et al., 1995; Lunt

et al., 1995; Zatz et al., 1995; Tawil et al., 1996; Hsu et al.,

1997; Ricci et al., 1999). In particular, larger deletions tend to

be associated with earlier onset and a more rapid progression

o FSHD (Goto et al., 1995; Lunt et al., 1995; Zatz et al., 1995;

Tawil et al., 1996; Hsu et al., 1997; Ricci et al., 1999). Impor-

tantly, it has been reported that at least one copy o D4Z4 is re-

quired to cause FSHD, as individuals with deletions o the entire

repeat array do not display signs o muscular dystrophy (Fig. 2;

Goto et al., 1995; Tupler et al., 1996; Rossi et al., 2007), sug-

gesting that the repeat itsel plays a critical role in the disease.

Figure 2. FSHD is caused by a reduction in the copy number of D4Z4 repeats. (A) Healthy individuals carry 11150 units o D4Z4. (B) FSHD patientshave less than 11 repeats. (C) At least one copy o D4Z4 is required or FSHD development, as individuals completely lacking D4Z4 are healthy.(D) Patients having a large deletion encompassing FRG2 and DUX4chave been described, indicating that these genes are not necessary or FSHD. Dottedlines indicate that distances are not to scale.



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long distance interactions and chromatin loop domain organiza-

tion (Maeda and Karch, 2007). Here, we summarize the studies

that have investigated the epigenetic actors involved in FSHD.

DNA methylation in FSHD. D4Z4 belongs to a

amily o human tandem repeats termed macrosatellites that are

noncentromerically located (Chadwick, 2009). Together with

other members o the amily, such as DXZ4 on chromosome X(Giacalone et al., 1992) and RS447 on 4p (Kogi et al., 1997),

D4Z4 is extremely GC rich.

DNA methylation is a chemical mark added to cytosine

residues by DNA methyltranserases: DNMT1, DNMT3A, and

DNMT3B (Chen and Li, 2004). Mammalian genomes are glob-

ally methylated, with the noticeable exception o short non-

methylated regions called CpG islands. Current data indicate

that promoter methylation leads to stable gene silencing, whereas

intragenic methylation helps to weaken transcriptional noise

(Suzuki and Bird, 2008). In addition, because several transcrip-

tion actors and chromatin-binding proteins, such as CTCF and

YY1, are methylation sensitive (Hark et al., 2000; Kim et al.,

2003), it is clear that DNA methylation can signicantly aect

the occupancy o a specic genomic region.

It has been shown that although D4Z4 is highly methyl-

ated in healthy subjects, FSHD patients have a specic hypo-

methylation o the D4Z4 contracted allele (Fig. 4; van Overveld

et al., 2003; de Gree et al., 2009). Recent ndings indicate that

D4Z4 contraction is always associated with hypomethylation,

irrespective o the chromosome or the haplotype, as deletions

on chromosome 10 and on chromosome 4 in asymptomatic car-

riers are also associated with a reduction in DNA methylation o

the contracted locus (de Gree et al., 2009). Moreover, D4Z4 is

was transerred to chromosome 10. Thus, the 4q35 FSHD can-

didate genes located proximal to the D4Z4 repeat array were

not present on chromosome 10. This nding suggested that

proximal 4q genes are not required or the pathogenesis o

FSHD (Lemmers et al., 2010b). It has to be noted, however, that

this case is a very unusual patient carrying a rare haplotype with

hybrid repeats deleted on 10q chromosome and a permissive4qA161 allele on 4q chromosome (Lemmers et al., 2010b).

Hence, in this case the disease could still be linked to chromo-

some 4 through an in trans eect o the hybrid 10q repeats on

the permissive chromosome 4q.

Although the exact molecular mechanism responsible or

the disease is unknown, it is agreed in the eld that the D4Z4

deletion causes an epigenetic gain-o-unction alteration lead-

ing to the up-regulation o candidate gene(s) (Neguembor and

Gabellini, 2010).

Epigenetic eatures associated with FSHD

Several o the clinical eatures outlined above, such as the gen-

der bias in severity, the asymmetric muscle wasting, and the dis-

cordance in monozygotic twins, suggest that FSHD development

involves epigenetic actors (Neguembor and Gabellini, 2010).

Epigenetic changes do not aect the primary DNA se-

quence; rather, gene expression is altered by changing the con-

ormation o chromatin. Local chromatin structure is regulated

by at least three processes: DNA methylation (Suzuki and Bird,

2008), histone modications (Ruthenburg et al., 2007), and

ATP-dependent chromatin remodeling (Ho and Crabtree, 2010).

In addition, a number o elements (chromatin boundaries, insu-

lators, etc.) aect higher order chromatin structure by regulating

Figure 3. SNPs and sequence variants at 4q35. 18 dierent 4q haplotypes have been described. FSHD patients carry D4Z4 deletions in 4qA161,4qA159, and 4qA168 backgrounds. These genetic contexts represent a permissive condition or the disease rather than a cause given that asymptomaticcarriers have been described. SSLP, simple sequence length polymorphism; TEL, telomere.



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ubiquitination, and SUMOylation o lysine (K) residues,

phosphorylation o serine (S) and threonine (T) residues, methyl-

ation o arginines (R), and ADP-ribosylation o glutamic acid

(Bernstein et al., 2007). Combinations o posttranslational

modications o single histones, single nucleosomes, and

nucleosomal domains establish local and global patterns o

chromatin modications and recruit nuclear actors that medi-

ate downstream unctions (Ruthenburg et al., 2007). These pat-

terns can be altered by multiple extracellular and intracellular

stimuli, and chromatin itsel unctions as a genomic integrator

o various signaling pathways, ultimately aecting cellular

processes such as replication and transcription (Cheung et al.,

2000; Nightingale et al., 2006).

The D4Z4 repeat array appears to be organized in distinct

domains, some characterized by transcriptionally repressive

heterochromatin and others by transcriptionally permissive

euchromatin (Zeng et al., 2009). In particular, on both chromo-

some 4 and 10, the repressive marks o histone H3 lysine 9

tri-methylation (H3K9me3) and histone H3 lysine 27 tri-

methylation (H3K27me3) are both present on some D4Z4 units,

but the permissive mark histone H3 lysine 4 di-methylation is

hypomethylated in patients aected by immunodeciency, cen-

tromeric instability, and acial anomalies syndrome (Kondo

et al., 2000). Interestingly, there are no common traits among

these diseases. These ndings suggest that D4Z4 hypomethyl-

ation is not responsible or FSHD by itsel. Nevertheless, it

is interesting to note that non4q-associated FSHD patients

(FSHD2), which lack D4Z4 contractions, display general D4Z4

hypomethylation on both chromosome 4 alleles and on the two

chromosome 10 alleles (de Gree et al., 2009), pointing to a

general deect in methylation o D4Z4 repeats. Collectively,

these results suggest that D4Z4 hypomethylation might repre-

sent a permissive condition required or FSHD onset or that it

might be a consequence o the primary cause o FSHD.

Histone modifcations in FSHD. Most cellular DNA

is compacted into nucleosomes, in which 146 bp o DNA are

wrapped around a protein octamer composed o two copies o

each core histone H2A, H2B, H3, and H4 (Campos and Reinberg,

2009). Nucleosomes are linked by a variable length o DNA

associated with linker histone H1 (Campos and Reinberg, 2009).

Histone proteins are subjected to dierent posttranslational

covalent modications, including acetylation, methylation,

Figure 4. Epigenetic features at 4q35 in healthy subjects and FSHD patients. In control individuals, the D4Z4 repeat array is characterized by markers ochromatin repression, such as high levels o DNA methylation, SUV39H1-mediated H3K9me3, and EZH2-mediated H3K27me3. In contrast, FSHD patientsdisplay hypomethylation, loss o H3K9me3, and corresponding loss o HP1 and cohesin binding. In healthy subjects, D4Z4 is specifcally bound by EZH2and a repressor complex composed o YY1, HMGB2, and nucleolin. It is still to be determined whether binding o these actors is altered in FSHD patients.The human 4q is perinuclear in both control and FSHD individuals. This localization depends on a region that is proximal to D4Z4 in healthy subjects butis D4Z4-specifc and CTCF-mediated in FSHD patients. A MAR located upstream o the repeat array was ound to be weakened in FSHD, altering the 3Dchromosomal architecture o the region.



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the 4q telomere is located near the nuclear periphery (Masny

et al., 2004; Tam et al., 2004). Interestingly, a sequence 215 kb

proximal to the repeat array shows a stronger localization to the

nuclear rim than D4Z4 in healthy subjects, suggesting that a re-

gion proximal to D4Z4, and not the repeat array itsel, directs

the 4q telomere to the periphery (Fig. 4; Masny et al., 2004).

Recently, Ottaviani et al. (2009b) identied an 80-bp sequence

inside the D4Z4 unit that can trigger perinuclear positioning o

articial telomeres in a CTCF- and lamin Adependent manner

(see below). This property is lost upon D4Z4 multimerization.

Thus, it appears that in healthy subjects, multiple copies o

D4Z4 are located near the nuclear periphery due to a 4q-specic

signal proximal to D4Z4, whereas in FSHD patients the peri-

nuclear location is mediated by D4Z4 (Fig. 4; Ottaviani et al.,

2009b). Although FISH analyses indicate that the peripheral

localization o 4q is maintained in dierent cell types and is

apparently unaltered in FSHD patients compared with controls,

the peripheral environment o the FSHD 4q35 allele may be

altered, and thereby contribute to the aberrant 4q35 gene ex-

pression reported in FSHD (Masny et al., 2004; Tam et al., 2004;

Ottaviani et al., 2009b).In metazoans, the nuclear lamina coats the inner surace o

the nuclear envelope (Hetzer, 2010). Using the DNA adenine

methyltranserase identication approach, lamin-associated do-

mains that correlate with silenced regions have been identied

(Guelen et al., 2008). Intriguingly, a lamin-associated domain has

been mapped to a locus that is 50 kb proximal to D4Z4, which is

consistent with the previous nding that this region has a role in

maintaining gene repression (Guelen et al., 2008). The peripheral

location o 4q seems to be strictly dependent on lamin A, given

that chromosome 4 telomeres are dispersed in cells lacking the

lamin A gene (Masny et al., 2004). Furthermore, chromatin

immunoprecipitation assays revealed that lamin A is associated

with D4Z4 in vivo (Ottaviani et al., 2009a).Altered chromatin organization at 4q35 in

FSHD. As mentioned above, there is no macroscopic relocal-

ization o 4q due to D4Z4 deletion in FSHD. Nonetheless,

more subtle alterations may occur. For example, the 4q35 locus

could be repositioned to a dierent peripheral subdomain, lead-

ing to inappropriate 4q35 gene regulation (Masny et al., 2004;

Ottaviani et al., 2009b). Alternatively, the higher order chroma-

tin structure o the 4q35 locus might be aected. There is grow-

ing evidence to indicate that the three-dimensional organization

o the FSHD region signicantly contributes to the regulation o

gene expression at 4q35 (Petrov et al., 2006; Pirozhkova et al.,

2008; Bodega et al., 2009).

Recently, it has been proposed that the area immediately

proximal to D4Z4 could play a role in FSHD (Lemmers et al.,

2007; Tsumagari et al., 2008). Interestingly, this area has been

suggested to unction as a nuclear matrix attachment region

(MAR; Petrov et al., 2006). Matrix attachment was shown to be

weakened on the contracted chromosome 4 in FSHD-derived

myoblasts compared with controls, leading to a drastic alter-

ation in chromatin loop domain organization (Fig. 4; Petrov

et al., 2006). In particular, whereas a high number o D4Z4 re-

peats maintain the organization o the repeat array and 4q35

genes in two distinct chromatin loops, loosening o the MAR in

present on dierent units (Zeng et al., 2009). Consistent with

previous studies, the authors reported euchromatin histone marks

in the rst proximal D4Z4 unit o the array (Jiang et al., 2003;

Zeng et al., 2009).

The modication o H3K9me3 on D4Z4 is mediated by

the histone methyltranserase SUV39H1 (Zeng et al., 2009).

Interestingly, H3K9me3 is lost in FSHD patients, preventing

the binding o D4Z4 to the heterochromatin-binding protein

HP1 and the sister chromatid cohesion complex, cohesin (Fig. 4).

This loss could lead to the de-repression o 4q35 genes and

muscular dystrophy (Zeng et al., 2009).

In a study aimed at characterizing the chromatin status o

the FSHD region, both D4Z4 and the promoter o the 4q35 gene

FRG1 (see below) were reported to be bound by the transcrip-

tion actor YY1 and the Polycomb Group protein EZH2 (Bodega

et al., 2009). Polycomb Group proteins are chromatin modiers

that implement transcriptional silencing in higher eukaryotes

(Simon and Kingston, 2009). In particular, YY1 and EZH2

binding are reduced both at D4Z4 and FRG1 promoter in myo-

tubes compared with myoblasts (Bodega et al., 2009). As a

consequence, the EZH2-mediated histone repressive markH3K27me3 is also reduced in myotubes compared with myo-

blasts. Accordingly, FRG1 expression is increased in myotubes

compared with myoblasts. Notably, the H3K27me3 modica-

tion at D4Z4 was ound by 3D FISH to be less abundant in

FSHD cells compared with controls (Bodega et al., 2009).

In summary, a number o repressive histone marks are

present on D4Z4 in healthy subjects and their loss in FSHD

might lead to de-repression o 4q35 genes.

A repressor complex binds to D4Z4. A ew years

ago, a 27-bp sequence located inside each D4Z4 unit, termed the

D4Z4 binding element, was identied (Gabellini et al., 2002).

This element is specically bound by a D4Z4 repressor com-

plex (DRC) composed o YY1, HMGB2, and nucleolin (Gabelliniet al., 2002). These actors interact with proteins that mediate

gene silencing and heterochromatin ormation, such as DNA

methyltranserases, histone deacetylases, and HP1 (Ko et al.,

2008; Wu et al., 2009). DRC binds to the 4q35 located D4Z4

in vivo and mediates the transcriptional repression o 4q35 genes

(Gabellini et al., 2002). Thus, the loss o D4Z4 repeats in FSHD

may result in reduced DRC binding to the region and, conse-

quently, reduced silencing o the 4q35 genes (Fig. 4; Gabellini

et al., 2004). Importantly, o the actors that have been identied

to bind D4Z4, YY1 is the only one with sequence specicity.

Thus, it would be interesting to investigate whether the recruit-

ment o actors like SUV39H1, EZH2, HP1, and cohesin to

D4Z4 is mediated by YY1 (Fig. 4).

Subnuclear localization o 4q35. Most nuclear

events do not occur randomly throughout the nucleoplasm;

rather, they are usually limited to specic and spatially dened

sites (Ferrai et al., 2010). Accordingly, the particular intra-

nuclear positioning o a given chromosomal region plays an

important role in several cellular processes, such as transcrip-

tion and replication (Spector, 2001).

Although mammalian telomeres in somatic cells are evenly

dispersed in the inner part o the nucleus (Ludrus et al., 1996;

Nagele et al., 2001; Amrichov et al., 2003; Weierich et al., 2003),



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The 4q35 locus is a relatively gene-poor region (van Geel

et al., 1999; Blair et al., 2002). O the genes that have been

identied at 4q35, those o particular interest areANT1, FRG1,

DUX4c, FRG2, andDUX4 (Li et al., 1989; van Deutekom et al.,

1996b; Gabrils et al., 1999; Rijkers et al., 2004; Bosnakovski

et al., 2008b). We will ocus here on the two main candidate

genes,DUX4 and FRG1. We will not discuss the other genes in

detail because they are less attractive candidates. For example,

DUX4c and FRG2 were ound to be deleted in some FSHD

amilies (Fig. 2; Lemmers et al., 2003), suggesting that they are

not necessary or disease onset. Nevertheless, these genes are

present in most o the aected amilies and it is possible that

they could contribute to the penetrance and severity o the disease.

There is some experimental support or this idea or DUX4c

(Bosnakovski et al., 2008b; Ansseau et al., 2009).

DUX4. Although theDUX4 gene contains an ORF en-

coding a putative double homeobox protein named DUX4, the

D4Z4 repeat was initially considered to be a nonprotein coding

sequence due to lack o evidence o transcription and protein

synthesis. Nonetheless, both DUX4 mRNA and protein were

recently detected in FSHD-derived primary myoblasts but notin controls, suggesting that D4Z4 may directly aect disease

progression through the aberrant production o DUX4 (Dixit

et al., 2007). Because the D4Z4 repeat does not contain a

canonical polyadenylation signal, the mRNA is generated exclu-

sively by transcription o the last, most distal, unit o the array

that extends to a region named pLAM, which contains a poly-

adenylation signal (Dixit et al., 2007). It was recently shown that

the pLAM polyadenylation signal can stabilize DUX4 tran-

scripts that are ectopically expressed in transected C2C12 cells

(Lemmers et al., 2010b). This pLAM sequence also contains a

polymorphism that could aect the polyadenylation o the dis-

talDUX4 transcript (Lemmers et al., 2010b). Because a group

o analyzed FSHD1 patients were all ound to carry the sameSNP in this region, it was suggested that this polymorphism

contributes to the selective stabilization oDUX4 transcripts in

FSHD (Lemmers et al., 2010b). It should be noted, however,

that in this study the permissive 4qA161 allele was compared only

to nonpermissive 4qB alleles or 10qA chromosomes (Lemmers

et al., 2010b). Non-permissive 4qA variants, such as the previ-

ously described 4qA166 (Lemmers et al., 2007; de Gree et al.,

2009), were not analyzed. Hence, the available data do not allow

us to exclude the possibility that the described variations refect

4qA/B or 4q/10q dierences.

TheDUX4 pre-mRNA can be alternatively spliced (Snider

et al., 2009). Interestingly, it was recently reported that muscles

o healthy subjects express low levels o aDUX4 splicing isoorm

encoding or a truncated protein, whereas muscles o FSHD pa-

tients express a splicing isoorm encoding or ull-length DUX4

(Snider et al., 2010). It has also been reported that the repeat array

displays a complex transcriptional prole that includes sense and

antisense transcripts and RNA processing (Snider et al., 2009).

Thus, there may be multiple D4Z4-derived RNA players in FSHD

and uture work will be required to determine their unctions.

Recently, an isogenetic screen to assess the eect o over-

expressing FSHD candidate genesANT1, FRG1, FRG2, DUX4c,

andDUX4 on cell viability was used (Bosnakovski et al., 2008a).

FSHD patients would bring the contracted repeats and 4q35

genes into the same chromatin loop (Petrov et al., 2006). Ulti-

mately, the presence o an enhancer at the 5 end o the D4Z4

unit could cause inappropriate 4q35 gene de-repression in FSHD

(Petrov et al., 2008).

Chromosome conormation capture (3C) is a technique

that identies long distance intra- and inter-chromosomal inter-

actions (Dekker et al., 2002). Using 3C, two groups have inde-

pendently investigated the higher order chromatin organization

o the 4q35 locus (Pirozhkova et al., 2008; Bodega et al., 2009).

Pirozhkova et al. (2008) showed that the telomeric 4qA allele

is in close proximity to the promoters o the 4q35 genes FRG1

and ANT1 in FSHD myoblasts and not in control myoblasts.

The 4qA allele is immediately distal to the D4Z4 repeat array

(Lemmers et al., 2002). Interestingly, an enhancer element was

detected in the 4qA allele that could be involved in the reported

up-regulation oFRG1 and ANT1 in FSHD (Gabellini et al.,

2002; Laoudj-Chenivesse et al., 2005; Pirozhkova et al., 2008).

In the other 3C study, an interaction between D4Z4 and the

FRG1 promoter was identied in human primary myoblasts that

appears to be highly reduced upon myogenic dierentiation(Bodega et al., 2009). Consistent with the observed mis-regulation

oFRG1, a small but statistically signicant reduction in the

D4Z4FRG1 promoter interaction was observed in FSHD myo-

blasts compared with controls (Bodega et al., 2009). Alto-

gether, it appears that in healthy subjects , the FRG1 promoter

is in close proximity with the D4Z4 repeat and the gene is

repressed, whereas in FSHD patients the promoter oFRG1

is in close proximity with the 4qA marker and the gene is up-

regulated (Gabellini et al., 2002; Pirozhkova et al., 2008; Bodega

et al., 2009).

CTCF is a multiunctional DNA-binding protein that is

important or transcriptional regulation, chromatin insula-

tion, and chromatin organization (Filippova, 2008). The same80-bp D4Z4 element mediating the perinuclear positioning

o the 4q telomere is also responsible or the CTCF and A-type

lamin-dependent transcriptional insulator unction o the re-

peat (Ottaviani et al., 2009a). The CTCF binding and insulation

activity are lost upon multimerization o the repeats (Ottaviani

et al., 2009a). As such, it has been proposed that FSHD patients

have a CTCF gain-o-unction phenotype that protects certain

genes rom the infuence o nearby repressive chromatin, ul-

timately generating a 4q35 de-repressed state (Fig. 4; Ottaviani

et al., 2009a).

Clearly, the FSHD locus is organized into a higher order

chromatin structure that undergoes dynamic remodeling. The

3D architecture o the region appears to play a undamental role

in regulating 4q35 chromatin status and gene expression, sug-

gesting that deects in the organization o the epigenome o the

FSHD region could underlie this disease.

FSHD candidate genes

The studies aimed at understanding the molecular basis o FSHD

indicate that an in cis alteration likely leads to the de-repression

o target gene(s). This model provides a valid explanation or

the 4q specicity and the autosomal-dominant transmission o

the disease (Tupler and Gabellini, 2004).



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proteins involved in RNA biogenesis, such as SMN, PABPN1, and

FAM71B (van Koningsbruggen et al., 2007). Interestingly, mu-

tations in SMNand PABPN1 cause myopathies (Calado et al.,

2000; Briese et al., 2005). Together, these results suggest that

FRG1 unctions in RNA processing. Indeed, in FSHD and in

transgenic mice or cells overexpressing FRG1, altered splicing

has been observed or a number o genes (Gabellini et al., 2006;

van Koningsbruggen et al., 2007; Davidovic et al., 2008).

Specic muscle-related unctions or FRG1 have been

identied in dierent animal models (Hanel et al., 2009; Liu

et al., 2010). Interestingly, overexpression oXenopusfrg1 or

C. elegans FRG-1 causes a muscle deect (Hanel et al., 2009;

Liu et al., 2010).

FRG1 contains a single ascin-like domain, a moti that is

associated with actin-bundling properties (Edwards and Bryan,

1995), and it was recently shown that FRG1 can bind F-actin

and promote its bundling (Liu et al., 2010). In agreement with

this nding, in dierent organisms the endogenous FRG1 in

muscle has not only a nuclear distribution but is also a sarco-

meric protein, suggesting that FRG1 might perorm a muscle-

specic unction (Hanel et al., 2010; Liu et al., 2010).FSHD pathology is most prominent in the musculature;

however, up to 75% o FSHD patients display retinal vasculopathy

(Fitzsimons et al., 1987; Padberg et al., 1995). Intriguingly, it

was recently reported that in human tissues the endogenous

FRG1 is strongly expressed in arteries, veins, and capillaries

(Hanel et al., 2010). Moreover, inXenopus FRG1 levels are

crucial or proper vascular development, and up-regulation

o FRG1 leads to a disrupted vascular phenotype (Wuebbles

et al., 2009).

Collectively, these studies indicate that FRG1 overexpres-

sion in dierent animal models is associated with aberrant mus-

cle structure and vasculature, the two most prominent eatures

o FSHD pathology.

Conclusions

Recent studies suggest that copy number variations (CNVs) are

important or human phenotypic diversity and disease suscepti-

bility. DNA repeats account or 55% o the human genome and

a signicant raction o CNVs.

FSHD is an important pathology caused by CNVs o

D4Z4 repeats. It is an extremely complicated and ascinating

disease, and research into this topic is revealing much about the

unctional organization o our genome.

An increasing amount o evidence suggests that the 4q35

macrosatellite repeat D4Z4 plays a crucial role in the chromo-

somal organization o the FSHD region. There is a general con-

sensus that the D4Z4 deletion in FSHD leads to epigenetic

alterations that aect the expression proles o genes within the

FSHD region. Unortunately, despite considerable eort, almost

20 years ater the identication o the genetic deect underlying

the disease, the causative FSHD gene(s) remains unknown, and

no eective treatments or FSHD are currently available.

The heterogeneity in disease maniestation probably re-

fects heterogeneity in gene expression in FSHD. An interesting

possibility, thereore, is that the complexity o FSHD could be

explained by considering it to be a contiguous gene syndrome,

Previous work demonstrated a pro-apoptotic unction or DUX4

(Kowaljow et al., 2007), and DUX4 overexpression was ound to

have a dramatically toxic eect. As a consequence o increased

DUX4 levels, the expression o oxidative stress response genes

and o myogenic actors (such as MyoD and My5) was altered.

Interestingly, a myogenic deect has been reported in FSHD

patients (Winokur et al., 2003a; Celegato et al., 2006).

DUX4 homeodomains are similar to those o Pax3 and

Pax7, which are transcription actors that are pivotal to muscle

unction (Buckingham, 2007). Additionally, the phenotype o

DUX4 overexpression can be rescued by overexpression o Pax3

or Pax7 (Bosnakovski et al., 2008a).

Recently, DUX4 overexpression (even at extremely low

levels) was reported to cause massive apoptosis and severely

abnormal development in Xenopus laevis, a model or verte-

brate development (Wuebbles et al., 2010). Note that an increase

in apoptosis is not generally considered to be a phenotype o

FSHD (Winokur et al., 2003b).

In theory,DUX4 represents an attractive FSHD candidate

gene, as it would easily explain the requirement or at least one

D4Z4 or the development o the disease. Due to its extremetoxicity, however, DUX4 could only unction in FSHD i it is ab-

sent under normal conditions and overexpressed exclusively in the

muscle cell precursors o FSHD patients (Wuebbles et al., 2010).

FRG1. FSHD region gene 1 (FRG1) is highly conserved in

both vertebrates and invertebrates (Grewal et al., 1998). FRG1

is considered a likely candidate gene or mediating FSHD due

to its selective chromosomal location at 4q35, its overexpres-

sion in FSHD samples (Gabellini et al., 2002; Bodega et al.,

2009), and the development o FSHD-like phenotypes ollow-

ing its overexpression in mice,Xenopus laevis, and Caenorhab-

ditis elegans (Gabellini et al., 2006; Hanel et al., 2009; Wuebbles

et al., 2009; Liu et al., 2010). Transgenic mice overexpressing

FRG1 selectively in the skeletal muscle develop pathologieswith physiological, histological, ultrastructural, and molecular

eatures that resemble those o FSHD patients (Gabellini et al.,

2006). Nevertheless, FRG1 overexpression in FSHD patients

is currently controversial, and studies have reported incon-

sistent results (Gabellini et al., 2002, 2006, Dixit et al., 2007,

Osborne et al., 2007; Bodega et al., 2009; Klooster et al., 2009).

The idea that FRG1 plays an important role in FSHD was very

recently challenged by the identication o an unusual FSHD

patient with deletion o D4Z4 only on chromosome 10 (Lemmers

et al., 2010b). However, as stated above, beore discarding a

causative role or FRG1 in FSHD, this patient requires urther

characterization. As discussed below, FRG1 is crucial or proper

muscle unction and vascular development (Gabellini et al.,

2006; Hanel et al., 2009; Wuebbles et al., 2009). Hence, FRG1

could at minimum aect the severity o the disease.

To better understand the role o FRG1 in FSHD, eorts

have been made to characterize its biological unction. The

human endogenous FRG1 protein copuries with the spliceo-

some, the proteinRNA macromolecular complex responsible

or pre-mRNA splicing (Kim et al., 2001; Rappsilber et al.,

2002; Bessonov et al., 2008). When ectopically overexpressed,

FRG1 localizes to nucleoli, Cajal bodies, and speckles (van

Koningsbruggen et al., 2004), and colocalizes and/or interacts with



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where the epigenetic alteration oDUX4, FRG1, and other

potential genes collaborate to determine the nal phenotype.

Finally, because DUX4 behaves as a transcriptional activator

(Dixit et al., 2007), it could play a direct role in transcriptional

overexpression o the other 4q35 genes, providing a uniying

model or the molecular mechanism o the disease.

Due to space limitations, we apologize to our colleagues or the vastly incom-

plete documentation o their important contributions to the topics describedin this review. We thank Drs. F. Jerey Dilworth, Claudia Ghigna, MichaelR. Green, and Lawrence G. Wrabetz or critical reading o the manuscript.

Research in the Gabellini laboratory is made possible by supportprovided by the European Research Council (ERC), the Muscular DystrophyAssociation o the USA (MDA), the European Alternative Splicing Networko Excellence (EURASNET), the Association Francaise contre les Myopathies(AFM), the FSHD Global Research Foundation, a Jaya Motta private donation,and the FSH Society, Inc. Davide Gabellini is a Dulbecco Telethon InstituteAssistant Scientist.

Submitted: 5 July 2010Accepted: 8 November 2010

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