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Published online before print
November 21, 2007, 10.1101/gr.6620908 Genome Res. 18:39-45, 2008 ©2008 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/08 $5.00
Letter A nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking activity in vivo: Implications for the facio-scapulo-humeral dystrophy1 UMR 8126, Centre National de la Recherche Scientifique–Université Paris-Sud 11, Institut de Cancérologie Gustave-Roussy, F-94804 Villejuif, France; 2 Institut National de la Santé et de la Recherche Médical ER125, F-34295 Montpellier, France
Facio-scapulo-humeral dystrophy (FSHD), a muscular hereditary disease with a prevalence of 1 in 20,000, is caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. Earlier, we demonstrated the existence in the vicinity of the D4Z4 repeat of a nuclear matrix attachment site, FR-MAR, efficient in normal human myoblasts and nonmuscular human cells but much weaker in muscle cells from FSHD patients. We now report that the D4Z4 repeat contains an exceptionally strong transcriptional enhancer at its 5'-end. This enhancer up-regulates transcription from the promoter of the neighboring FRG1 gene. However, an enhancer blocking activity was found present in FR-MAR that in vitro could protect transcription from the enhancer activity of the D4Z4 array. In vivo, transcription from the FRG1 and FRG2 genes could be down- or up-regulated depending on whether or not FR-MAR is associated with the nuclear matrix. We propose a model for an etiological role of the delocalization of FR-MAR in the genesis of FSHD.
Facio-scapulo-humeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease with a prevalence of 1 in 20,000 (Lunt and Harper 1991  ). FSHD is characterized by progressive weakness and atrophy of muscles of the face, upper arms, and shoulder girdle. The disorder is associated with a shortened repeat array that remains present at a subtelomeric position on chromosome 4q after deletion of an integral number of 3.3-kb tandem repeats. The size of the D4Z4 polymorphic locus varies in normal individuals from 35 to 300 kb but is consistently less than 35 kb in length in FSHD patients (van Deutekom et al. 1993). D4Z4 elements have been shown to contain a cryptic DUX4 gene potentially coding for a double homeodomain protein (van Geel et al. 1999). An overall perturbation of mRNA expression profiles and protein content has been observed in FSHD patients (Tupler et al. 1999; Winokur et al. 2003; Wohlgemuth et al. 2003; Laoudj-Chenivesse et al. 2005). However, since no gene has been found altered by the intrachromosomal deletion, the mechanism leading to FSHD remains unexplained and alternative hypotheses are clearly needed.
In eukaryotic nuclei and metaphase chromosomes, the DNA is organized into loop domains (for review, see Vassetzky et al. 2000
Previously, we demonstrated the existence of a nuclear matrix attachment site (S/MAR) in the immediate vicinity of the D4Z4 repeat (Fig. 1). We then demonstrated that the S/MAR adjacent to the D4Z4 array was prominent in normal human myoblasts and nonmuscular human cells but much weaker in muscle cells derived from FSHD patients. We also reported that the D4Z4 repeat array and upstream genes reside in a single loop in FSHD myoblasts but are located in two distinct loops in nonmuscular cells and normal human myoblasts (Petrov et al. 2006
Here, we have studied in detail the ability of the D4Z4 repeat to regulate transcriptional activity. We have found evidence of an exceptionally strong transcriptional enhancer at the 5'-end of the repeat. This enhancer could up-regulate transcription from the FRG1 gene promoter. We have also found that the S/MAR located in the vicinity of the D4Z4 array exerts an enhancer blocking activity in vivo, thus insulating the FRG1 and FRG2 genes from the effect of D4Z4. We propose a model whereby this S/MAR regulates chromatin accessibility and expression of genes implicated in the genesis of FSHD.
The D4Z4 repeat contains a strong enhancer A partial deletion of the D4Z4 repeat array on human chromosome 4q affects transcription of neighboring genes (Tupler et al. 1999 ; Winokur et al. 2003; Wohlgemuth et al. 2003; Laoudj-Chenivesse et al. 2005). To test the hypothesis that the repeat array could directly regulate gene transcription, we tested the transcription modulating activity of a D4Z4 repeat unit using a pGL3 luciferase reporter system. Constructs and control plasmids were transfected into human primary myoblasts where reporter gene expression was measured 48 h post-transfection. As seen in Figure 1B, the presence of the SV40 enhancer increased transcription fourfold in comparison with the enhancer-less pGL3-promoter plasmid. The D4Z4 repeat cloned into the pGL3–promoter plasmid stimulated luciferase synthesis with efficiency similar to that observed with the SV40 enhancer used as a positive control. Thus, the D4Z4 repeat exhibits properties of a transcriptional enhancer.
Transcriptional activators, or enhancers, are short DNA sequences composed of elements ranging in size from 10 to several hundreds of nucleotide pairs that promote transcription irrespective of their location in relation with the regulated gene. Enhancers can act at very long distances, greater than 100 kb. They can be located either at the 5'-end or 3'-end of coding sequences as well as within introns. To characterize the D4Z4 enhancer, we submitted a repeat unit to progressive deletion. This translated into an increasing enhancing effect. The strongest transcriptional enhancing activity was obtained with the two smallest fragments produced, which were 319 and 170 bp in length. Located at the 5'-end of the D4Z4 repeat, these two fragments increased transcription 12-fold as compared with the basal promoter and proved to be threefold more powerful than the SV40 enhancer (Fig. 1B). The increased activity of the minimal enhancer as compared with the whole D4Z4 repeat unit may be explained by the presence of a transcriptional repressor element, D4Z4 binding element (DBE) (Gabellini et al. 2002 We then tested the tissue and species specificity of the minimal 170-bp-long enhancer by transfecting reporter plasmids into the murine myoblast cell line C2C12, the human rhabdomyosarcoma (RMS) cell line, and HeLa cells. Similar to the SV40 enhancer, this minimal D4Z4 enhancer was found to lack any strong tissue or species specificity (Fig. 1C). When further tested in stable transfectants, the enhancer produced similar results (data not shown).
The D4Z4 enhancer up-regulates transcription from the FRG1 promoter
FR-MAR exhibits an enhancer-blocking activity in vivo Earlier studies have shown that gene expression profiles in the vicinity of the D4Z4 array are different in normal versus FSHD myoblasts (Tupler et al. 1999 ; Winokur et al. 2003; Wohlgemuth et al. 2003; Laoudj-Chenivesse et al. 2005). This may be the result of the reduction of the D4Z4 array and subsequent changes in chromatin organization (for review, see van der Maarel et al. 2006). Interestingly, it has been suggested that the D4Z4 repeat unit could possess both repressor and enhancer activities (Gabellini et al. 2002; Petrov et al. 2003; Rijkers et al. 2004). We have recently reported that the D4Z4 array was separated from neighboring genes by a nuclear matrix attachment site (FSHD-related S/MAR or FR-MAR), which does not function in the context of the retracted D4Z4 array in FSHD primary myoblasts (Petrov et al. 2006). Since S/MARs can increase gene transcription via an insulator-like effect (for review, see Bode et al. 2000), we decided to test the hypothesis that FR-MAR could similarly serve as an insulator protecting genes from the effect of neighbor sequences. A series of pNEO plasmids were constructed based on the pGL3 series where the luciferase reporter gene was replaced by a neomycin resistance gene. First, the above-characterized 170-bp D4Z4 minimal enhancer was inserted in place of the SV40 enhancer in the pNEO plasmid. After transfection into HeLa cells and selection in the presence of G418, the number of resistant colonies was counted. As expected, the number of resistant colonies increased as compared with controls transfected with the enhancer-less plasmid (Fig. 3B). The FR-MAR and adjacent sequences (a 1263-bp HindIII fragment excised from the pGEM42 plasmid, see Fig. 3A) were then inserted, resulting in the number of G418-resistant colonies being reduced from 52 ± 7 to 22 ± 5 (Fig. 3B, cf. pNeo01 and pNeo02). As a further control, the 368-bp FR-MAR-containing fragment was then deleted from pNeo03 by HincII deletion and religation. This reconstituted the original ability of colonies to grow in the presence of G418 (45 ± 3 colonies), thus reinforcing the notion that FR-MAR indeed confers enhancer-blocking activity to transgenes (Fig. 3B, pNeo03). However, similar results would also have been obtained if FR-MAR acted as a silencer. To test this hypothesis, we used pNeo04 where the FR-MAR and adjacent sequences had been inserted in a plasmid containing only the SV40 promoter. Here, no inhibitory effect was obtained as compared with basal transcription (pNeo04, 15 ± 1 colonies vs. 17 ± 5 colonies for pNeo05, Fig. 3B). In our conditions, the construct integration efficacy was similar for all constructs, with an average of 1.5 copies per genome (Fig. 3C). The neomycin gene transcription has also been verified by Northern blot, and the data were very similar to those found by the colony counting method (Fig. 3D). From these data, we conclude that the FR-MAR indeed has enhancer-blocking activity.
These results appeared somewhat surprising in the light of the generally accepted notion that S/MARs augment the level of transcription of integrated transgenic constructs by insulating them from a generally unfavorable chromatin environment (Bode et al. 2000 ). In the present situation, FR-MAR seemed to act differently since it did not protect the construct from an inhibitory environment (compare pNeo04 and pNeo05) but rather blocked the activity of adjacent enhancers. It is known that the S/MAR activities are highly context-dependent (Schubeler et al. 1997). While boundary functions are mostly provided by extended S/MAR elements (Kalos and Fournier 1995), short elements show a spectrum of activities depending on the system The 368-bp FR-MAR may simply be too short to provide boundary functions. A similar situation could well occur in FSHD myoblasts. Indeed, we have previously shown that, while FR-MAR is associated with the nuclear matrix in normal myoblasts, in myoblasts from FSHD patients, its counterpart present on the deleted chromosome 4q is delocalized from the nuclear matrix.
FR-MAR containing constructs are associated with the nuclear matrix In this assay, we evaluated the frequency of matrix attachment of the FR-MAR present in pNeo01 and pNeo04 constructions. As seen in Figure 4 and as expected, no association to the matrix was observed when the negative control oligonucleotide was used. In contrast, the positive MYC probe was found bound to the matrix in all transfectants. Similarly, the FR-MAR probe was found consistently bound to the matrix. To exclude that this FR-MAR association to the matrix was due to the native (genomic) FR-MAR, a DNA fragment adjacent to the FR-MAR in the constructs was used as a probe (pNeo). Similar to the adjacent FR-MAR, this probe associated with the matrix, thus confirming that the FR-MAR in the constructs behaves as does its genomic counterpart.
Overall, the data we have obtained can be summarized as schematized in Figure 5. In normal cells (Fig. 5, top panel), the FR-MAR that lies between the centromeric FRG1/FRG2 genes and the telomeric array of D4Z4 repeats is attached to the nuclear matrix, physically separating and functionally protecting the FRG genes from the regulatory activity of the strong transcriptional enhancer present in each D4Z4 repeat unit. In contrast, in deleted myoblasts from FSHD patients (Fig. 5, bottom panel), the FR-MAR is dissociated from the nuclear matrix because of the chromosomal deletion on its telomeric side (Petrov et al. 2006 ). Transcription of FRG1/FRG2 genes is accordingly activated by the enhancer elements present in the remaining repeats. Whether and how this phenomenon could play a role in the genesis of FSHD will require further studies.
The mechanism of dissociation of the FR-MAR from the nuclear matrix in FSHD cells remains unknown; one of the hypotheses is based on the observation that a partial loss of D4Z4 repeat array in FSHD patients is linked to hypomethylation of the D4Z4 array and adjacent sequences (van Overveld et al. 2003 ; de Greef et al. 2007). It is known that some proteins that mediate specific association of DNA with the nuclear matrix, for example, MECP2 (Stratling and Yu 1999; Horike et al. 2005), only interact with the methylated DNA. Thus, interaction with the hypomethylated FR-MAR may be lost in FSHD patients. We are currently exploring this hypothesis and searching for the protein factor(s) that are involved in interaction with the FR-MAR in normal cells and myoblasts from the FSHD patients. Whether and how this phenomenon could play a role in the genesis of FSHD will require further studies.
Cell lines C2C12 and HeLa cell line was purchased from American Type Culture Collection and grown in the Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal calf serum. RMS cells were a kind gift of Dr. S. Leibowitz.
Vectors and cloning A series of pNEO vectors was used for stable transfection studies. The pNeo02 vector was obtained by inserting the neomycin resistance cassette from the pIRES vector (Clontech), in place of the Luciferase unit in the pGL3-Pro-170 vector. This vector contains the 170-bp D4Z4 minimal enhancer upstream of the Luciferase promoter. The vectors were digested by BamHI and HindIII.
The D4Z4 repeat and the FR-MAR were obtained from the pGEM42 plasmid containing two D4Z4 repeats and the flanking sequences (Gabriels et al. 1999 The FR-MAR was excised from the 1263-bp fragment by HincII digestion and religated to construct the pNeo03 vector. pNeo04 and pNeo05 were obtained by excising the 170-bp D4Z4 minimal enhancer by MluI digestion from the pNeo01 and pNeo03 vectors, respectively.
Transient transfection and reporter gene assays The constructs (with the firefly luciferase gene) were cotransfected with an internal control (with a Renilla luciferase gene) in each case. Dual-Luciferase Reporter Assay System (Promega) was used for the reporter gene expression assay. In this assay, the activities of firefly (construct) and Renilla (control) luciferases are measured sequentially from a single sample using the Top count NXT fluorimeter (Hewlett-Packard).
Stable transfection
Determination of the number of integrated pNeo constructs
Nuclei and nuclear matrices Nuclear matrices were prepared by treatment of the isolated nuclei with NaCl as follows: Digestion buffer (100 mM NaCl, 25 mM KCl, 10 mM Tris-HCl at pH 7.5, 0.25 mM spermidine) was added to 105 nuclei to a final volume of 400 µl. DNase I was added to a final concentration of 100 µg/mL, and the samples were digested for 2 h at 4°C, followed by the addition of CuCl2 to a final concentration of 1 mM for 10 min at 4°C. The nuclei were then extracted by addition of one volume of a buffer containing 4 M NaCl, 20 mM EDTA, and 40 mM Tris-HCl at pH 7.5. The resulting nuclear matrices were spun in a microfuge at 2000g for 10 min at 4°C and then washed three times with a buffer containing 2 M NaCl, 10 mM EDTA, and 20 mM Tris-HCl at pH 7.5. Nuclear matrices were digested with proteinase K and extracted with phenol-chloroform. The obtained nuclear matrix-associated DNA was treated with RNase A and either radioactively labeled using the Ready-to-Go kit (APBiotech) or labeled with DIG using DIG-High Prime Kit (Roche) and used as a probe for hybridization.
We thank Dr. A. Belayev for her kind gift of the pGEM42 plasmid. We thank Ms. Ingrid Lenglart for her help in some experiments. The research has been supported by grants from the Association Française contre les Myopathies (AFM) to Y.S.V. and D.L. A.P. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale. I.P. was supported by a post-doctoral fellowship from AFM.
3 These authors contributed equally to this work. 
E-mail vassetzky{at}igr.fr; fax 33 1 42 11 54 94. Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.6620908
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Received April 17, 2007; accepted in revised format October 16, 2007.  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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Abstract
Results and Discussion
caffold/
atrix-
ssociated 
egions (Mirkovitch et al. 1984
-globin gene domain using oligonucleotide DNA arrays. Genomics 85: 143–151.