Skip Navigation


Human Molecular Genetics Advance Access originally published online on September 30, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/22/2895    most recent
ddg327v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (46)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Winokur, S. T.
Right arrow Articles by Flanigan, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winokur, S. T.
Right arrow Articles by Flanigan, K. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 22 2895-2907
DOI: 10.1093/hmg/ddg327
© 2003 Oxford University Press

Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation

Sara T. Winokur1,*, Yi-Wen Chen2, Peter S. Masny1, Jorge H. Martin1, Jeffrey T. Ehmsen1,{dagger}, Stephen J. Tapscott3, Silvere M. van der Maarel4, Yukiko Hayashi5 and Kevin M. Flanigan6

1Department of Biological Chemistry, University of California, Irvine, CA, USA, 2Children's National Medical Center, Washington, DC, USA, 3Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 4Leiden University Medical Center, The Netherlands, 5National Institute for Neuroscience, Tokyo, Japan and 6Eccles Institute of Genetics, University of Utah, Salt Lake City, UT, USA

Received June 9, 2003; Accepted September 18, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) results from integral deletions of the subtelomeric repeat D4Z4 on chromosome 4q. A disruption of chromatin structure affecting gene expression is thought to underlie the pathophysiology. The global gene expression profiling of mature muscle tissue presented here provides the first insight into an FSHD-specific defect in myogenic differentiation. FSHD expression profiles generated by oligonucleotide microarrays were compared with those from normal muscle as well as other types of muscular dystrophies (DMD, aSGD) in order to determine FSHD-specific changes. In addition, matched biopsies (affected and unaffected muscle) from individuals with FSHD served to monitor expression changes during the progression of the disease as well as to diminish non-specific changes resulting from individual variability. Among genes altered in an FSHD-specific and highly significant manner, many are involved in myogenic differentiation and suggest a partial block in the normal differentiation program. Indeed, many of the transcripts affected in FSHD represent direct targets of the transcription factor MyoD. Additional mis-expressed genes confirm a diminished capacity to buffer oxidative stress, as demonstrated in FSHD myoblasts. This enhanced vulnerability of proliferative stage myoblasts to reactive oxygen species is also disease-specific, further implicating a defect in FSHD muscle satellite cells. Importantly, none of the genes localizing to the FSHD region at 4q35 were found to exhibit a significantly altered pattern of expression in FSHD muscle. This finding was corroborated by expression analysis of FSHD muscle using a custom cDNA microarray containing 51 genes and ESTs from the 4q35 region. Disruptions in FSHD myogenesis and oxidative capacity may therefore not arise from a position effect mechanism as has been previously suggested, but rather from a global effect on gene regulation. Improper nuclear localization of 4qter is discussed as an alternative model for FSHD gene regulation and pathogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder involving a characteristic pattern of muscles from which it derives its name (1,2). Facial weakness is frequently the earliest sign of the disease and is almost invariably present. Progression to shoulder girdle musculature often prompts the initial clinical evaluation. Asymmetric involvement of affected musculature is highly characteristic of FSHD, with the biceps often displaying considerable weakness, while there is a relative sparing of the deltoids. The pectoralis may be altogether absent. The basis of this asymmetry and the unique pattern of affected musculature are unknown. Extra-muscular manifestations of FSHD may include sensorineural hearing loss and retinal telangiectasias, with epilepsy and mental retardation in the most severe cases (3,4). Inflammation of muscle can be a prominent histologic feature (5).

FSHD is inherited as an autosomal dominant trait, although as many as 10–30% of cases have no family history and are the result of de novo mutations (6,7). Age of onset typically is in the second decade of life, with nearly complete penetrance (95%) by age 20 (8). The molecular rearrangement associated with clinical development of FSHD is a deletion within a large polymorphic EcoRI fragment located in the subtelomeric region of the long arm of chromosome 4 (9). A ‘short’ EcoRI fragment (<38 kb) containing the probe locus p13E11 segregates with FSHD, while the size of this polymorphic locus in the normal population ranges between 38 and 300 kb. The FSHD associated DNA rearrangement is due to deletions of integral copies of a 3.3 kb tandem repeat unit termed D4Z4 contained within this EcoRI fragment (10). Fine mapping of this region reveals that the 4q subtelomere is mainly characterized by this large polymorphic repeat array (D4Z4) located ~25 kb proximal to the telomeric TTAGGG repeat (11–13). D4Z4 contains internal LSau and 68 bp Sau3A repeats as well as a putative open reading frame encoding two homeodomain sequences (DUX4) (14). However, despite intense efforts in many laboratories over the past decade, no protein coding transcripts have been identified from the D4Z4 repeat sequence (1). All characterized genes in this region map proximal to the 3.3 kb repeats (12).

Thus, FSHD results from a highly unusual mechanism in which the mutation does not reside within the gene(s) responsible for the disease. The position effect hypothesis has been invoked to explain the relationship of D4Z4 repeat deletions and the onset of FSHD (15,16). The D4Z4 repeat has several characteristics of heterochromatin, the highly condensed chromosomal structure often responsible for gene silencing (11,13,15,17–20). Although direct evidence of D4Z4 heterochromatization is lacking, the position effect hypothesis proposes that deletions of D4Z4 allow for a local decondensation of chromatin, with consequent de-repression of gene expression in the FSHD region. A multiprotein complex that binds to D4Z4 heterochromatin in vivo is proposed to negatively regulate gene expression, with D4Z4 deletions in FSHD permitting increased transcription at 4q35 (21).

A previous study seems to support the position effect hypothesis in FSHD, by claiming that several genes on 4q35 (FRG1, FGR2 and ANT1) are upregulated in FSHD muscle (21). While these results have yet to be replicated, it is worth noting that non-quantitative RT–PCR was used in this study. Recent analysis of these same transcripts using real-time quantitative RT–PCR has not detected upregulation of these genes in skeletal muscle from FSHD patients (M. Ehrlich and S. van der Maarel, personal communication). In addition, numerous other studies have not demonstrated altered gene expression at 4q35 (22,23). Thus, much skepticism exists regarding a possible position effect mechanism as the basis for FSHD.

The current study utilizes the GeneChip oligonucleotide microarray platform, a sensitive and specific means of identifying differentially expressed transcripts, to examine FSHD gene expression both at 4q35 as well as on a genome-wide scale (24). The majority of genes found to be dysregulated in FSHD are involved in myogenic differentiation and cell-cycle control. A custom cDNA microarray of all presently characterized 4q35 genes and ESTs was also generated in order to directly test the position effect hypothesis for FSHD. We report here that both oligonucleotide and cDNA microarray analysis do not reveal any evidence for increased expression of 4q35 genes in FSHD muscle. An alternate model for disease pathogenesis involving inappropriate nuclear localization of the FSHD chromosomal region is proposed to explain the genome-wide disruption of genes involved in myogenic differentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSHD gene expression profiles
Using the criteria described under data analysis (minimum 2-fold change and statistically significant at P<0.05) in three FSHD patients, a total of 230 genes were found to be dysregulated, representing 3.5% of the transcripts arrayed on the HuFL GeneChip. In order to expand the number of transcripts queried and the statistical power of FSHD expression data, a larger set of biopsies (nine FSHD and six normal) was evaluated using a second GeneChip format (U95A). Utilizing the same significance criteria, a total of 297 genes (2.6%) were found to be dysregulated in FSHD muscle. Not surprisingly, a number of genes with altered expression in FSHD are also dysregulated in other forms of muscular dystrophy. Of 131 genes altered in Duchenne and a-sarcolglycan muscular dystrophy (25) using the identical GeneChip format, 35 (27%) are also altered in FSHD. All of the significant expression changes were determined using regularized t-test analysis within a Bayesian statistical framework, allowing for a robust estimate of variance (see Materials and Methods). Raw expression data, as well as statistical analyses of the data, are posted as supplementary data at www.ucihs.uci.edu/biochem/winokur under ‘publications’.

Of the FSHD-specific dysregulated genes, a large number have a role in cellular proliferation and differentiation (Table 1). The common functional role of many of these genes is most evident in the overlap between HuFL and U95A data. Table 1 lists FSHD dysregulated genes that meet inclusion criteria for both sets of microarray data (2-fold change and P<0.05 on both the HuFL and U95A GeneChips). For any particular gene, the presence on both of these ‘significant gene’ lists increases the confidence that that gene is involved in FSHD. The complete list of all significantly altered genes from both the HuFl and U95A GeneChips is found as supplementary data on the website www.ucihs.uci.edu/biochem/winokur.


View this table:
[in this window]
[in a new window]
  		Table 1. Significantly altered genes in FSHD
 
More than half of the significantly altered genes in FSHD muscle are involved in cell-cycle control, proliferation and differentiation. Cysteine and glycine-rich protein 3 (CSRP3), also known as muscle LIM protein (MLP), is increased nearly 3-fold, whereas this transcript is not altered in the other types of muscular dystrophy studied. CSRP3 functions as a positive regulator of myogenesis (26). The LIM/double zinc-finger motif found in CSRP3 is present in proteins with essential functions in gene regulation, cell growth and somatic differentiation. Another gene involved in the differentiation of many cell types and upregulated in FSHD muscle is the delta-like homolog DLK1 (27). WEE1, a Cdk-inhibitory kinase that functions in part to arrest the cell cycle (28), is elevated in FSHD. The transferrin receptor TFRC is also elevated in FSHD muscle. Transferrin is a key myoblast trophic factor, initially promoting myoblast proliferation and subsequently supporting myogenic differentiation (29). Two other genes, very low density lipoprotein receptor (VLDLR) and necdin (NDN), which play important roles in differentiation, are upregulated in FSHD muscle. VLDLR is required for neurogenesis of the cerebral cortex (30) and necdin is an imprinted gene that suppresses growth in postmitotic neurons (31).

Other genes of interest in light of FSHD pathophysiology include immunoglobulin lambda-like polypeptide 2 (IGLL2) and thioredeoxin interacting protein TXNIP (also known as vitamin D3 up-regulated protein 1, VDUP1). As FSHD muscle often has marked inflammatory infiltrates (5), identifying specific genes involved such as IGLL2 may prove valuable in dissecting the role of the immune response in this disease. TXNIP functions as an oxidative stress mediator by inhibiting activity of thioredoxin, which is a potent thiol reductase and reactive oxygen species regulator (32). TXNIP is down-regulated in FSHD muscle, a finding consistent with the enhanced vulnerability to oxidative stress seen in FSHD myoblasts (33). Interestingly, a number of metallothionein (MT) transcripts are also reduced in FSHD muscle. Metallothioneins are a group of ubiquitous low MW proteins that have functional roles in cell growth, repair and differentiation. MTs serve to protect against free radical toxicity during the differentiation of myoblasts to myotubes (34).

Independent confirmation of CSRP3/MLP and DLK1 upregulation
In order to independently verify expression changes of a small subset of genes involved in differentiation (CSPR3 and DLK1), real time RT–PCR (TaqMan) analysis was carried out (35). Owing to limiting RNA isolated from muscle biopsies used for GeneChip analysis, only a few samples and primer sets were used for confirmation. A negative control was chosen which did not demonstrate altered expression according to microarray analysis. Actinin-associated LIM protein (ALP) maps to the FSHD gene region and is not altered in FSHD (22,36). Expression levels for all transcripts were determined relative to the internal housekeeping control gene ribonucleoprotein S1 (RPNS1) (37). As Figure 1 demonstrates, both CSPR3 and DLK1 were significantly increased upon real-time RT-PCR analysis, while no difference in ALP levels was detected.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. TaqMan (real-time PCR) analysis confirms the alteration of specific transcripts in FSHD muscle. PCR primers were generated for representative transcripts (MLP, DLK and ALP) expressed in muscle. Both MLP and DLK demonstrate increased expression in FSHD muscle (P-value <0.006 and <0.027, respectively), verifying corresponding GeneChip data. ALP is not altered in FSHD muscle, confirming the expression pattern on the U95A GeneChip.

 
Expression changes in common with other muscular dystrophies
FSHD expression profiles were also compared to those generated for other muscular dystrophies using the identical GeneChip format (25). Several of the significantly dysregulated genes in FSHD muscle were also found to be affected in other types of muscular dystrophy (DMD, {alpha}SGD; Table 2). It is interesting to note that a large percentage of these genes is probably dysregulated as a result of fibrotic infiltration. These genes include such extracellular matrix proteins as collagen types I and VI, lumican, fibronectin and tenascin. Other categories of genes affected in common with other types of muscular dystrophy include immune response genes such as complement and coagulation factors, and genes involved in energy metabolism.


View this table:
[in this window]
[in a new window]
  		Table 2. Common dysregulated genes in FSHD, DMD and {alpha}-SGD
 
FSHD-specific genes that are involved in progression of the disease can be identified by comparing clinically affected versus unaffected muscles within single dystrophy patients
Matched pairs of muscle biopsies from affected and unaffected muscle within the same patient are an extremely valuable resource to monitor the progression of disease. Expression profiling of three sets of paired muscle samples (affected and unaffected muscle from each of three individuals) was performed on the HuFL GeneChip. Expression profiling of affected to unaffected muscle highlights the genes that correlate with progressive dystrophy. Table 3 lists the genes that are deregulated in affected FSHD muscle (greater than 50% of the 12 pairwise comparisons). Of the 12 genes, six (50%) represent a further dysregulation of genes involved in the dystrophic process, as these same genes are altered in other muscular dystrophies. The deregulation of these transcripts is likely to be a secondary effect that contributes to the symptoms or compensatory response in FSHD (as they correlate with progressive dystrophy), but not the early primary cause of the disease. The genes affecting cellular differentiation (CSRP3, TFRC, DLK1 and WEE1, listed in Table 1) are not further increased, and thus are likely to be related to the primary defect in FSHD. However, as FSHD affects specific muscle groups more severely than others, the possibility that primary changes are actually more pronounced in the affected muscle cannot be ruled out.


View this table:
[in this window]
[in a new window]
  		Table 3. Comparison analysis of affected to unaffected FSHD muscle
 
GeneChip expression analysis does not support a 4q35 position effect
Expression of 4q35 genes in FSHD muscle is summarized in Table 4. Eight FSHD region (4q35) genes are present on both the HuFL and U95A arrays, with an additional seven genes present on the U95A GeneChip. Using the same criteria (>2-fold change in expression, P<0.05) we found that none of the genes in the 4q35 region are significantly altered. We find no elevation of FRG1 in this study, although are not able to discern 4q35-specific transcripts of this multi-copy gene as the hybridization-based approach used in this study allows only for the expression analysis of all FRG1-like sequences in the genome. However, for all single-copy genes in the 4q35 FSHD region (FACL2, ANT1, IRF2, KLKB1, FAT, CASP3, F11, TLR3, ALP, ING1L, ARGBP2, DCTD and DKF2P564J102), none exhibit a significantly altered pattern of expression in FSHD upon oligonucleotide microarray analysis.


View this table:
[in this window]
[in a new window]
  		Table 4. Oligonucleotide (GeneChip) microarray analysis of 4q35 gene expression
 
cDNA microarray analysis
A cDNA microarray was constructed containing amplicons for 51 genes and ESTs localized to 4q35 and 26 genes and ESTs localized to 10q26, a region containing homologous D4Z4-like subtelomeric repeats. Six FSHD and five normal total muscle RNA samples hybridized independently against the identical internal reference control RNA sample using the constructed microarrays. Results were statistically analyzed to evaluate changes in expression level.

Importantly, we find neither a significant misregulation of 4q35 genes nor a gradient of expression throughout the 4q35 gene region. Relative gene expression levels in FSHD versus normal muscle for all of the characterized genes in 4q35 are listed in Table 5. Fold-changes (FC) range from 0.7 to 1.6 for 4q35 genes, and 0.5–1.9 for the housekeeping genes. Several amplicons from genes found to be dysregulated on the GeneChip (CSRP3, limatin and lumican) were also dysregulated on the cDNA array (3.5–9.5 FC), verifying the potential of this custom array to detect differences in expression levels. All of the cDNA microarray data, including 4q35, 10q26, housekeeping and dysregulated genes and expression values for each of the FSHD and normal muscle samples, can be found as supplementary data on the website www.ucihs.uci.edu/biochem/winokur.


View this table:
[in this window]
[in a new window]
  		Table 5. cDNA microarray analysis of 4q35 gene expression
 
Relative expression levels throughout the 4q35 region are depicted in Figure 2, with closed circles representing characterized, named genes and the open circles corresponding to ESTs. The size of each circle reflects significance of observed fold change (smaller P-value corresponds to larger circle). Clearly, a gradient does not exist correlating relative expression of 4q35 genes in FSHD versus normal muscle with distance from the telomere. This again is contradictory to the suggestion of a FSHD position effect presented by Gabellini et al. (21).



View larger version (7K):
[in this window]
[in a new window]
  		Figure 2. Fold change of 4q35 transcript expression levels in FSHD versus normal muscle using cDNA microarray analysis. Neither a significant misregulation of 4q35 genes nor a gradient of expression throughout 4q35 is noted. Size of each circle reflects significance of observed fold change (smaller P-value corresponds to larger circle). Closed circles are characterized, named genes. Open circles correspond to ESTs. The 4q telomere is represented by the far right of the graph.

 
Many FSHD dysregulated genes are direct targets of MyoD
Many of the genes with altered expression patterns in FSHD muscle are early direct targets of MyoD (38). Of the 63 characterized genes identified as direct MyoD targets in primary murine fibroblasts transfected with a MyoD-ER fusion construct, 46 are also represented on the HuFL and U95A arrays used in this study. Of these 46 genes, 11 (24%) are significantly altered in FSHD (Table 6A). In addition to these direct targets of MyoD, many other genes identified in this study directly influence the activity of MyoD and the process of myogenic differentiation. Table 6B lists all significantly altered MyoD target genes from either the HuFL or U95A GeneChips. Each of these genes displayed a greater than 2-fold change in expression level and a P-value of <0.05. The complete list of all significantly altered genes, amongst which are the MyoD target genes listed in Table 6B, can be found at www.ucihs.uci.edu/biochem/winokur.


View this table:
[in this window]
[in a new window]
  		Table 6. FSHD altered genes that are (A) direct targets of MyoD or (B) involved in myogenic differentiation
 
Muscle LIM protein (CSRP3/MLP) is significantly upregulated in FSHD muscle (2.9-fold, P=6.9x10-7 on HuFL array, 2.4-fold, P=2.5x10-5 on U95A array). CSRP3/MLP is a positive regulator of myogenesis, promoting myogenic differentiation through enhancing the activity of MyoD (39). Overexpression of MLP in C2 myoblasts induces myogenic differentiation (26). Another gene with increased expression in FSHD that directly interacts with MyoD is the MyoD family inhibitor MDF1 (40). Four and a half LIM domains 2 (FHL2), another gene involved in myoblast differentiation, is also up-regulated in FSHD muscle. FHL2 is down-regulated during transformation of normal myoblasts to rhabdomyosarcoma cells (41). FHL2 interacts with insulin growth factor receptor 5 (42), a direct target of MyoD also altered in FSHD (38).

Delta-like homolog DLK1, a gene involved in the differentiation of several cell types (43), is also elevated in FSHD. Increased expression of DLK1 has been shown to be responsible for the muscular hypertrophy in callipyge sheep (44) and may be responsible for the abdominal muscular abnormalities in paternal UPD14 (45). The muscular hypertrophy in callipyge sheep is probably due to the inhibitory affect of DLK on differentiation. Delta indirectly regulates the transcription of myogenic target genes such as myocyte enhancer factor 2C (MEF2C) (46). MEF2C is down-regulated in FSHD muscle (Table 5) (47). MEF2C interacts with HDAC4, key regulator of myogenesis, also down-regulated in FSHD muscle (48). HDAC4 itself is regulated through calcium/calmodulin signaling, and may be affected by decrease in calmodulin RNA in FSHD muscle (Table 1).

Other genes found to be dysregulated in FSHD that play key roles in differentiation and accompanying apoptosis include members of the transforming growth factor, caspase and metallothionein families (Table 6B). Myogenesis requires the coordinate regulation of cell cycle withdrawal and enhanced apoptosis (49). Metallothioneins are recruited into the nucleus to protect against free-radical toxicity, oxidative stress and apoptosis (50,51). Both caspase 1 and 2 are elevated in FSHD muscle (Table 6B), while a large number of metallothionein genes are reduced in expression. Indeed, FSHD myoblasts demonstrate an increased vulnerability to oxidative stress (33), which may influence the ability of these cells form fully functional myotubes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The gene expression profiling data presented here suggests an intriguing hypothesis for the pathophysiological basis of FSHD: a primary defect in muscle cell differentiation. Many of the genes dysregulated in FSHD are involved in myogenesis, cellular differentiation and cell-cycle control. Aberrant transcription of genes involved in differentiation and proliferation could either result from an intrinsic defect in FSHD muscle, or result from enhanced regeneration and recruitment of satellite cells in affected muscle. As the majority of these genes are deregulated in an FSHD disease-specific manner, and are not dysregulated in the other muscular dystrophies studied, we propose that some of these expression differences result from a primary differentiation defect in FSHD muscle rather than a result of secondary dystrophic changes. Although the mechanism of gene dysregulation in FSHD remains unknown, the genes mis-expressed in FSHD are highly enriched for genes that interact with, or are direct targets of, MyoD, suggesting an enhancement of an early sub-program of MyoD-mediated muscle cell differentiation.

In support of this hypothesis, FSHD myoblasts in culture are seen to fuse at a faster rate than controls, suggesting that the muscle differentiation program has been turned on at an earlier, perhaps premature timepoint (D. Figlewicz, personal communication) (52). This may be mediated by the up-regulation of cyclin dependent kinase inhibitor protein, p21, in FSHD myoblasts (33,52), also a direct target of MyoD (53). The premature fusion of FSHD myoblasts is not due to replicative senescence of these cells in diseased muscle, as telomeric/centromeric repeat ratios do not differ between FSHD and normal myoblasts (52).

As FSHD is an adult-onset muscular dystrophy, one must also consider the possibility that myogenesis in FSHD results from inefficient termination of the myogenic program once differentiation has occurred. The continued expression of genes involved in differentiation, as seen in this study, may interfere with proper function of muscle in the differentiated state. Interestingly, alteration in D4Z4 copy number has been shown to affect myogenic differentiation in C2C12 myoblasts (54). Deformed myotube morphology and a reduced myotube fusion index result from increasing numbers of D4Z4. While disease is inversely correlated to the number of D4Z4 repeats in FSHD, the demonstration that D4Z4 can affect myogenesis in trans is further support for a disruption of this process in FSHD.

Although a gradient of altered expression throughout 4q35 has been proposed as part of a position effect model for FSHD (21), our data firmly disputes this. Both oligonucleotide and cDNA microarray analysis do not reveal any evidence for increased expression of 4q35 genes in FSHD muscle. Examination of all single-copy genes on 4q35 (FACL2, ANT1, IRF2, KLKB1, FAT, CPP32, F11, TLR3, ALP, ING1L, ARGBP2, HPGD, DCTD and MTNR1A) does not reveal an aberrant pattern of gene expression. This conclusion is supported by several other studies of 4q35 gene expression. Bouji et al. (22) examined the single copy 4q35 gene actinin-associated LIM protein (ALP) in FSHD and normal muscle. No difference in either RNA or protein expression levels of ALP was observed. Van Deutekom et al. (23) investigated allele-specific FRG1 steady-state transcript levels using RNA-based single-strand conformation polymorphism (SSCP) analysis. No evidence for a position effect on allelic transcription was obtained in lymphocytes or muscle biopsies from patients and controls. Recent analysis of the single copy 4q35 gene adenine nucleotide translocator (ANT1) as well as FRG1 expression using real-time RT–PCR again did not detect up-regulation of these transcripts in FSHD skeletal muscle tissue (M. Ehrlich and S. van der Maarel, personal communication). All of these studies on 4q35 gene expression, as well as the data we present here, have reached a different conclusion than that reported by Gabellini et al. (21). The reason for this discrepancy is as yet unclear.

As the vast majority of expression studies do not support the position effect model for FSHD, we propose an alternate model for FSHD pathogenesis. Alterations in 4q35 subtelomeric chromatin structure may affect the global expression of genes involved in myogenic differentiation rather than a regional disruption of 4q35 genes. Recent data lends strong support to consideration of FSHD as a chromatin disease. A polymorphism of the 4q telomere exists in the population with nearly equal frequencies (12). FSHD is uniquely associated with the 4qA allele variant containing ß-satellite, a sequence previously associated with heterochromatin (55). In addition, many D4Z4 CpG methylation sensitive restriction sites are significantly hypomethylated in FSHD patients compared to normal individuals (S. van der Maarel, personal communication) (56).

However, the differences in chromatin structure are unlikely to act in cis (i.e. from a localized position effect), as 4q35 gene expression does not appear to be altered in FSHD muscle. Instead, we propose that FSHD may result from improper nuclear localization. D4Z4 deletions may affect the localization of 4qtel to a nuclear domain in which aberrant gene expression can occur. Appropriate nuclear localization is essential for normal gene expression (57). Telomeric regions are known to localize to discrete nuclear domains and regulate expression of genes within this domain (58,59). As the 4q telomere is one of very few telomeres to localize to the nuclear periphery in myoblasts (our unpublished data), the role of D4Z4 in attachment to the nuclear envelope is an area of active research. Disruption of the nuclear envelope in other forms of neuromuscular disease, such as Emery–Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD1B), dilated cardiomyopathy (CMD1A) and autosomal recessive Charcot–Marie–Tooth disease (AR-CMT2) is well established (60,61). Emerin and lamin A/C mutations underlie these disorders, and are thought to be pathogenic either through direct effects on nuclear envelope integrity or through perturbations in tissue-specific gene expression patterns (62). Effects on gene expression may arise through inappropriate methylation status of sequences normally attached to the nuclear envelope or matrix (63,64). Defective DNA replication and cell cycle progression, cellular functions also intimately involved with the nuclear envelope and lamina, may lead to the increased vulnerability to free radical damage seen in FSHD myoblasts.

While precise definition of pathogenetic mechanism responsible for FSHD remains elusive, this study brings together many of the disparate finding on FSHD: altered rates of myoblast fusion and cell cycle progression, enhanced oxidative stress and differential methylation. Many of these findings can now be seen as either the cause or effect of alterations in specific stages of myogenic differentiation. As the specific gene expression changes likely to be responsible for this aberration are not seen in other types of muscular dystrophies, we propose that FSHD exhibits a unique defect in myogenic differentiation. This defect may be related to global effects on gene expression mediated by nuclear localization and alterations in nuclear envelope association rather than a postion effect on 4q35 specific gene expression. Future directions in FSHD should therefore focus on more global analysis of cellular processes such as the cell cycle and the effects of nuclear localization on gene expression of muscle transcripts, and not solely on the identification and analysis of 4q35 specific genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Muscle biopsies
Initially, expression profiling was performed on three sets of matched muscle biopsies (affected and unaffected muscles from three independent FSHD patients) and three normal controls using the HuFL GeneChip. Additional biopsy sets (nine FSHD and six normal controls) were then analyzed on the U95A GeneChip in order to extend and confirm the dysregulation of specific transcripts identified by the HuFL GeneChip. Gene expression profiles were generated comparing both FSHD affected muscle to normal muscle (HuFL and U95A GeneChips), as well as FSHD affected muscle to unaffected muscle (biceps and deltoid, respectively) within three patients (HuFL GeneChips). In order to reduce noise due to experimental variability, target cRNA from each biopsy was prepared in replicate, and independently hybridized to one of two HuFL GeneChips for that biopsy sample. In order to reduce noise due to individual variability, and to monitor the progression of muscle involvement in FSHD, matched biopsies from individual FSHD patients were used. Thus, a total of nine biopsies were prepared in replicate and hybridized to 18 HuFL GeneChips. The expression profiles were then compared to a series of disease control profiles including Duchenne muscular dystrophy and {alpha}-sarcoglycan deficiency (25) using identical RNA isolation methodology and GeneChip formats. No corresponding dataset exists for the FSHD versus normal dataset, although a larger set of muscle (nine FSHD and six normal) were used for the analysis.

cRNA preparation, hybridization and staining.
Muscle biopsies were used to extract total RNA by using TRIzol® reagent (GibcoBRL Life Technologies, MD, USA). Each biopsy was divided into two fragments, and RNA isolated from each fragment independently. Ten micrograms of total RNA from each biopsy fragment were converted into double stranded cDNA by using SuperScript Choice system (GibcoBRL Life Technologies, MD, USA) with an oligo-dT primer containing T7 RNA polymerase promoter (Integrated DNA Technologies, Coralville, IA, USA). The double-stranded cDNA was purified with phase lock gel 1 light (5 Prime, Boulder, CO, USA), then used for in vitro transcription using the MEGAscript T7 IVT kit (Ambion). Biotin-labeled cRNA was purified by RNeasy kit (Qiagen, CA, USA), and fragmented randomly to approximately 200 bp (200 mM Tris–acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc). Fifteen micrograms of fragmented cRNA were hybridized to Affymetrix GeneChip HuFL or U95A microarrays for 16 h. The microarray was washed and stained by the Affymetrix Fluidics Station 400, using instructions and reagents provided by Affymetrix. This involves removal of non-hybridized probe, and then incubation with phycoerythrin-streptavidin to detect bound probe. Fluorescent images were read using the Hewlett-Packard G2500A Gene Array Scanner.

Data analysis
Raw expression data, as well as statistical analyses of the data, are posted on the website www.ucihs.uci.edu/biochem/winokur. Analysis of hybridization intensities for the HuFL and U95A array was performed with GeneChip software Version 3.3 and Version 4, respectively, developed by Affymetrix. Briefly, each gene is queried by perfect-match (PM) probes as well as mis-match (MM) probes with a centrally placed single base change. Comparison of the hybridization signals from the PM and MM probes allows a measure of signal intensity the average difference (AD), and elimination of most non-specific cross-hybridization from the data analysis. Values of intensity difference as well as ratios of each probe pair are used for determination of whether a gene is called ‘present’ or ‘absent’. Quality control (QC) criteria and data for each array, such as internal and hybridization controls, percentage present calls and oligo B2 corner patterns, are posted on the website listed above.

For the comparison of FSHD and normal muscle expression data, AD values for each transcript across all muscle samples were imported into Excel, and output was then analyzed with Cyber T. Cyber T is a statistical program based upon Student's t-test which is designed for output data from large-scale microarray experiments (65). Regularized t-tests between experimental groups are generated within a Bayesian statistical framework (66). Estimates of variance of expression levels for each gene are therefore improved by including the variance of other genes with similar expression levels. Transcripts were considered to be significantly dysregulated if they displayed a 2-fold or greater fold change and had a Cyber T generated P-value <=0.05.

For the comparison of FSHD affected versus unaffected muscle, rather than utilize AD values across all samples, we were interested in comparing expression profiles within patients. Thus, each affected sample was prepared in replicate and then compared to each replicate from the unaffected muscle for each patient, yielding a total of 12 pairwise comparisons. For the comparative analysis, a difference call (DC) was made for each transcript when comparing two samples. This DC value was based upon the difference in AD value, as well as the number of probe pairs that either increased or decreased in intensity (increase or decrease ratio). Transcripts were considered significantly dysregulated if they displayed consistent DCs in >40% of all pairwise comparisons and had greater an average fold change of greater than 1.5 across all pairwise comparisons. Such criteria were chosen in part to allow for cross-species comparison of FSHD dysregulated genes to a list of direct MyoD targets (38). The DC were then assigned a numerical value (I=1, NC=0, D=-1) for each pairwise comparison. Transcripts were then sorted in Excel according to how consistently they were dysregulated across all FSHD versus normal comparisons by calculating the sum of difference calls (SDC). Expression data from each experiment, as well as complete comparative analyses represented as excel files, and statistical analyses of the data, are posted on the website (http://www.ucihs.uci.edu/biochem/winokur).

Taqman (real-time PCR)
Independent confirmation of select expression changes was performed by real-time RT–PCR (TaqMan) analysis (36). Primers and probes were designed using Primer Express software (PE Applied Biosystems), targeting the terminal 3' intron/exon boundary of each transcript to reduce genomic contamination signals (Table 7).


View this table:
[in this window]
[in a new window]
  		Table 7. TaqMan RT-PCR primers and probes used for expression confirmation
 
cDNA syntheses, including no-amplification controls, were performed in triplicate using the TaqMan RT Reagents (PE Applied Biosystems), with 1 µg of muscle biopsy total RNA for each 100 µl reaction. TaqMan PCR assays for each amplicon were performed in duplicate in 96-well optical plates on cDNA equivalent to 10 ng of total RNA. Typical 50 µl reactions contained 17 µl dH2O, 25 µl 2x Taqman Universal PCR Master Mix (PE Applied Biosystems), 1 µl sense primer (20 uM), 1 µl antisense primer (10 µM), and 1 µl Taqman probe (5 µm), and 5 µl cDNA. Thermal cycling conditions were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Standard curves for each amplicon were generated using serial dilutions of known quantities of reverse-transcribed skeletal muscle total RNA (Stratagene). Data was collected using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems), and relative quantities of initial template for each cDNA sample were extrapolated from the standard curve based on threshold cycle (Ct) values.

Construction of cDNA microarrays
A custom glass slide cDNA array consisting of 107 transcripts was made in order to test the position effect hypothesis for FSHD. The cDNAs included 51 genes and expressed sequence tags (ESTs) mapped to 4q35, 26 genes and ESTs mapped to 10q26, 12 housekeeping genes (37) and 18 miscellaneous genes known to be causative for other types of muscular dystrophy or dysregulated according to FSHD muscle GeneChip analysis. The selection of 4q35 and 10q26 ESTs was based on known regional genes and exhaustive Unigene (http:Unigene) database searches to include as complete a set of ESTs as possible at the time the array was constructed. cDNA clones (IMAGE consortium, Research Genetics), preferably with inserts about 1 kb in length and near the 3' end of the cDNA, were amplified with vector primers and column resin purified. For those genes unavailable from the IMAGE consortium or for which clonal amplification failed, appropriate primers were made based on the known gene sequence, and the product was PCR amplified from cDNA library templates. All amplicons were sequence-verified with a PRISM 377 DNA Sequencer (Applied Biosystems) and examined by agarose gel electrophoresis and spectrophotometry to insure the quality and identity of the products. The cDNA amplicons were diluted to 1 µg/µl in spotting solution M1435 (Sigma) and arrayed onto CMT-GAPS slides (Corning) using an OmniGrid arrayer (GeneMachines). The spot diameter was 120 µm. Each cDNA was represented in duplicate on the microarray, and the housekeeping genes were represented four times each. After plotting, slides were baked at 80°C for 3 h.

Probe hybridization
Labeling was carried out using the NEN Micromax TSA Labeling and Detection Kit (Perkin Elmer Life Sciences, Boston, MA, USA). RT–PCR was performed with 2 µg of total RNA using the fluorescein nucleotide reaction mix. For all slides, RT–PCR was also performed on 2 µg of Universal RNA (Stratagene) using the biotin reaction mix for use as an internal reference control on each slide. RT–PCR was performed for 2 h at 42°C. cDNA was isolated by isopropanol precipitation and resuspended together in TSA kit hybridization buffer Q. All slides were prehybridized by heating in a 5xSSC, 0.1% SDS, 0.1% BSA solution at 42°C for 45 min, then rinsed in water and isopropanol. The hybridization mixture was denatured and then hybridized to the slides overnight in a 64°C oven in a hydrated hybridization chamber (Corning Life Sciences). Slides were washed and incubated with anti-Fl-HRP followed by Cy3-tyramide to deposit Cy3. They were then washed and incubated with streptavidin-HRP followed by Cy5-tyramide to deposit Cy5.

cDNA microarray detection and quantification
Slides were scanned using a Scanarray 4000 XL scanner (GSI Lumonics) using a 550 nm laser for Cy3 and 649 nm for Cy5. Multiple scans were made for each slide using increasing laser intensities, in order to include bright signals which quickly saturate the scanner as well as weak signals which only appear at high laser intensities. The images were quantified using the Dapple program (http:Dapple), which provides both a reading of the signal intensity of a spot and a judgment of the quality of the identified spot. Spots having a very low intensity or intensity near the scanner's maximum were excluded. Only spots simultaneously deemed acceptable on both the Cy3 and Cy5 image were used. A Cy3/Cy5 intensity ratio was calculated for each accepted spot pair. For each scan of a slide, the spot intensity ratios were normalized by dividing the measured intensity ratios by the median of the measured housekeeping gene intensity ratios of that scan. The log value of this ratio was calculated to produce a normalized log ratio (NLR). A single NLR value for each spot on the slide was made by taking the median of the acceptable NLRs on all scans of the spot (spots appearing on less than two of the scans were rejected). A single NLR was assigned to each transcript on a slide by taking the median of values for redundant spots of that transcript. Finally, FSHD slides were compared to the normal slides. Within groups, only transcripts for which at least half different slides provided a NLR value were included. The median NLR for each transcript within the FSHD group was divided by the corresponding median value within the normal group, providing a fold change of the transcript expression level. Confidence was evaluated using the Wilcoxon rank sum test.


   ACKNOWLEDGEMENTS
 
Our deepest gratitude goes to the now deceased Dr Kiichi Arahata for his enthusiasm, expertise and collaborative spirit. His presence is greatly missed by all members of the FSHD community. We wish to thank Drs Ko Sahashi (Aichi Medical University) and Masanori Nakagawa (Kyoto Prefectural University of Medicine) for their contribution of muscle biopsy specimens for this study. This study was funded by grants from the MDA (S.T.W.), FSH Society, Inc. (S.T.W. and K.M.F.) and NIH (S.T.W., Y.-W.C.). Additional support was generously provided by the FischerShaw Foundation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Biological Chemistry, 202 Sprague Hall, University of California, Irvine, CA 92697, USA. Tel: +1 9498242750; Fax: +1 9498249547; Email: stwinoku{at}uci.edu Back

{dagger} Present address:

Johns Hopkins University School of Medicine, Baltimore, MD, USA. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tawil, R., Figlewicz, D.A., Griggs, R.C. and Weiffenbach, B. (1998) Facioscapulohumeral dystrophy: a distinct regional myopathy with a novel molecular pathogenesis. Ann. Neurol., 43, 279–282.[CrossRef][Web of Science][Medline]

  2. Padberg, G. and Adams, C. (2000) Facioscapulohumeral muscular dystrophy. In Pulst, S.-M. (ed.), Neurogenetics. Oxford University Press, Oxford, pp. 105–116.

  3. Padberg, G.W., Brouwer, O.F., de Keizer, R.J.W., Gruter, A.M., Wijmenga, C., Grote, J.J. and Frants, R.R. (1992) Retinal vascular disease and sensorineural deafness are part of facioscapulohumeral muscular dystrophy. Am. J. Hum. Genet., 51 (S), A104.

  4. Funakoshi, M., Goto, K. and Arahata, K. (1998) Epilepsy and mental retardation in a subset of early onset 4q35-facioscapulohumeral muscular dystrophy. Neurology, 50, 1791–1794.[Abstract/Free Full Text]

  5. Arahata, K., Ishihara, T., Fukunaga, H., Orimo, S., Lee, J.H., Goto, K. and Nonaka, I. (1995) Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immuno-cytochemical and genetic analyses. Muscle Nerve, 2, S56–66.[CrossRef][Medline]

  6. Padberg, G.W., Frants, R.R., Brouwer, O.F., Wijmenga, C., Bakker, E. and Sandkuijl, L.A. (1995) Facioscapulohumeral muscular dystrophy in the Dutch population. Muscle Nerve, 2, S81–84.[Medline]

  7. Zatz, M., Marie, S.K., Passos-Bueno, M.R., Vainzof, M., Campiotto, S., Cerqueira, A., Wijmenga, C., Padberg, G. and Frants. R.R. (1995) High proportion of new mutations and possible anticipation in Brazilian facioscapulohumeral muscular dystrophy families. Am. J. Hum. Genet., 56, 99–105.[Web of Science][Medline]

  8. Lunt, P.W., Compston, D.A. and Harper, P.S. (1989) Estimation of age dependent penetrance in facioscapulohumeral muscular dystrophy by minimising ascertainment bias. J. Med. Genet., 26, 755–760.[Abstract/Free Full Text]

  9. Wijmenga, C., Hewitt, J.E., Sandkuijl, L.A., Clark, L.N., Wright, T.J., Dauwerse, H.G., Gruter, A.M., Hofker, M.H., Moerer, P., Williamson, R. and Frants, R.R. (1992) Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy. Nat. Genet., 2, 26–30.[CrossRef][Web of Science][Medline]

  10. van Deutekom, J.T., Wijmenga, C., van Tienhoven, E.A.E., Gruter, A.-M., Hewitt, J.E., Padberg, G.W., van Ommen, G.-J.B., Hofker, M.H. and Frants, R.R. (1993) FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum. Mol. Genet., 2, 2037–2042.[Abstract/Free Full Text]

  11. Hewitt, J.E., Lyle, R., Clark, L.N., Valleley, E.M., Wright, T.J., Wijmenga, C., van Deutekom, J.C., Francis, F., Sharpe, P.T., Hofker, M. et al. (1994) Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystrophy. Hum. Mol. Genet., 3, 1287–1295.[Abstract/Free Full Text]

  12. van Geel, M., Dickson, M.C., Beck, A.F., Bolland, D.J., Frants, R.R., van der Maarel, S.M., de Jong, P.J. and Hewitt, J.E. (2002) Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin. Genomics, 79, 210–217.[CrossRef][Web of Science][Medline]

  13. Bengtsson, U., Altherr, M.R., Wasmuth, J.J. and Winokur, S.T. (1994) High resolution fluorescence in situ hybridization to linearly extended DNA visually maps a tandem repeat associated with facioscapulohumeral muscular dystrophy immediately adjacent to the telomere of 4q. Hum. Mol. Genet., 3, 1801–1805.[Abstract/Free Full Text]

  14. Gabriels, J., Beckers, M.C., Ding, H., De Vriese, A., Plaisance, S., van der Maarel, S.M., Padberg, G.W., Frants, R.R., Hewitt, J.E., Collen, D. and Belayew, A. (1999) Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element. Gene, 236, 25–32.[CrossRef][Web of Science][Medline]

  15. Winokur, S.T., Bengtsson, U., Feddersen, J., Mathews, K.D., Weiffenbach, B., Bailey, H., Markovich, R.P., Murray, J.C., Wasmuth, J.J., Altherr, M.R. and Schutte, B.C. (1994) The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease. Chromosome Res., 2, 225–234.[CrossRef][Medline]

  16. Lyle, R., Wright, T.J., Clark, L.N. and Hewitt, J.E. (1995) The FSHD-associated repeat, D4Z4, is a member of a dispersed family of homeobox-containing repeats, subsets of which are clustered on the short arms of the acrocentric chromosomes. Genomics, 28, 389–397.[CrossRef][Web of Science][Medline]

  17. Qumsiyeh, M.B. (1999) Structure and function of the nucleus: anatomy and physiology of chromatin. Cell Mol. Life Sci., 8, 1129–1140.

  18. Parseghian, M.H., Newcomb, R.L., Winokur, S.T. and Hamkalo, B.A. (2000) The distribution of somatic H1 subtypes is nonrandom on active vs inactive chromatin. Chromosome Res., 8, 405–424.[CrossRef][Web of Science][Medline]

  19. Tsien, F., Sun, B., Hopkins, N.E., Vedanarayanan, V., Figlewicz, D.A., Winokur, S.T. and Ehrlich, M. (2000) Hypermethylation of the FSHD syndrome-associated D4Z4 repeat in normal and FSHD somatic cell populations but not in ICF syndrome cells. Mol. Genet. Metab., 74, 322–331.

  20. Lemmers, R.J., van der Maarel, S.M., van Deutekom, J.C., van der Wielen, M.J., Deidda, G., Dauwerse, H.G., Hewitt, J., Hofker, M., Bakker, E., Padberg, G.W. and Frants, R.R. (1998) Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis. Hum. Mol. Genet., 7, 1207–1214.[Abstract/Free Full Text]

  21. Gabellini, D., Green, M.R. and Tupler, R. (2002) Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell, 10, 339–348.

  22. Bouju, S., Pietu, G., Le Cunff, M., Cros, N., Malzac, P., Pellissier, J.F., Pons, F., Leger, J.J., Auffray, C. and Dechesne, C.A. (1999) Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohumeral muscular dystrophy (FSHD) candidate genes. Neuromusc. Disord., 9, 3–10.[CrossRef][Web of Science][Medline]

  23. van Deutekom, J.C., Lemmers, R.J., Grewal, P.K., van Geel, M., Romberg, S., Dauwerse, H.G., Wright, T.J., Padberg, G.W., Hofker, M.H., Hewitt, J.E. and Frants, R.R. (1996) Identification of the first gene (FRG1) from the FSHD region on human chromosome 4q35. Hum. Mol. Genet., 5, 581–590.[Abstract/Free Full Text]

  24. Lipshutz, R.J., Fodor, S.P., Gingeras, T.R. and Lockhart, D.J. (1999) High density synthetic oligonucleotide arrays. Nat. Genet., 21, 20–24.[CrossRef][Web of Science][Medline]

  25. Chen, Y.-W., Zhao, P., Borup, R. and Hoffman, E.P. (2000) Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J. Cell Biol., 151, 1321–1336.[Abstract/Free Full Text]

  26. Arber, S., Halder, G. and Caroni, P. (1994) Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell, 79, 221–231.[CrossRef][Web of Science][Medline]

  27. Laborda, J. (2000) The role of the epidermal growth factor-like protein dlk in cell differentiation. Histol. Histopath., 15, 119–129.[Web of Science][Medline]

  28. Igarashi, M., Nagata, A., Jinno, S., Suto, K. and Okayama, H. (1991) Wee1(+)-like gene in human cells. Nature, 353, 80–83.[CrossRef][Medline]

  29. Chen, G. and Quinn, L.S. (1992) Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract. J. Cell. Physiol., 153, 563–574.[CrossRef][Web of Science][Medline]

  30. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R.E., Richardson, J.A. and Herz, J. (1999) Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell, 97, 689–701.[CrossRef][Web of Science][Medline]

  31. Nakada, Y., Taniura, H., Uetsuki, T., Inazawa, J. and Yoshikawa, K. (1998) The human chromosomal gene for necdin, a neuronal growth suppressor, in the Prader–Willi syndrome deletion region. Gene, 213, 65–72.[CrossRef][Web of Science][Medline]

  32. Junn, E., Han, S.H., Im, J.Y., Yang, Y., Cho, E.W., Um, H.D., Kim, D.K., Lee, K.W., Han, P.L., Rhee, S.G. and Choi, I. (2000) Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function. J. Immunol., 164, 6287–6295.[Abstract/Free Full Text]

  33. Winokur, S.T., Barrett, K., Martin, J.H., Forrester, J.R., Simon, M., Tawil, R., Chung, S.-A., Masny, P.S. and Figlewicz, D.A. (2003) Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress. Neuromusc. Disord., 13, 322–333.[CrossRef][Web of Science][Medline]

  34. Apostolova, M.D., Ivanova, I.A. and Cherian, M.G. (1999) Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity. Toxicol. Appl. Pharmac., 159, 175–184.

  35. Medhurst, A.D., Harrison, D.C., Read, S.J., Campbell, C.A., Robbins, M.J. and Pangalos, M.N. (2000) The use of TaqMan RT–PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J. Neurosci. Meth., 98, 9–20.[CrossRef][Web of Science][Medline]

  36. Xia, H., Winokur, S.T., Kuo, W.L., Altherr, M.R. and Bredt, D.S. (1997) Actinin-associated LIM protein: identification of a domain interaction between PDZ spectrin-like repeat motifs. J. Cell Biol., 139, 507–515.[Abstract/Free Full Text]

  37. Warrington, J.A., Nair, A., Mahadevappa, M. and Tsyganskaya, M. (2000) Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol. Genomics., 2, 143–157.[Abstract/Free Full Text]

  38. Bergstrom, D.A., Penn, B.H., Strand, A., Perry, R.L., Rudnicki, M.A. and Tapscott, S.J. (2002) Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol. Cell, 9, 587–600.[CrossRef][Web of Science][Medline]

  39. Kong, Y., Flick, M.J., Kudla, A.J. and Konieczny, S.F. (1997) Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol. Cell. Biol., 17, 4750–4760.[Abstract]

  40. Chen, C.M., Kraut, N., Groudine, M. and Weintraub, H. (1996) I-mf, a novel myogenic repressor, interacts with members of the MyoD family. Cell, 86, 731–741.[CrossRef][Web of Science][Medline]

  41. Genini, M., Schwalbe, P., Scholl, F.A., Remppis, A., Mattei, M.-G. and Schafer, B.W. (1997) Subtractive cloning and characterization of DRAL, a novel LIM-domain protein down-regulated in rhabdomyosarcoma. DNA Cell Biol., 16, 433–442.[Web of Science][Medline]

  42. Amaar, Y.G., Thompson, G.R., Linkhart, T.A., Chen, S.T., Baylink, D.J. and Mohan, S. (2002) Insulin-like growth factor-binding protein 5 (IGFBP-5) interacts with a four and a half LIM protein 2 (FHL2). J. Biol. Chem., 277, 12053–12060.[Abstract/Free Full Text]

  43. Ruiz-Hidalgo, M.J., Gubina, E., Tull, L., Baladron, V. and Laborda, J. (2002) dlk modulates mitogen-activated protein kinase signaling to allow or prevent differentiation. Exp. Cell Res., 274, 178–188.[CrossRef][Web of Science][Medline]

  44. Charlier, C., Segers, K., Karim, L., Shay, T., Gyapay, G., Cockett, N. and Georges, M. (2001) The callipyge mutation enhances the expression of coregulated imprinted genes in cis without affecting their imprinting status. Nat. Genet., 27, 367–369.[CrossRef][Web of Science][Medline]

  45. Kurosawa, K., Sasaki, H., Sato, Y., Yamanaka, M., Shimizu, M., Ito, Y., Okuyama, T., Matsuo, M, Imaizumi, K., Kuroki, Y. and Nishimura, G. (2002) Paternal UPD14 is responsible for a distinctive malformation complex. Am. J. Med. Genet., 110, 268–272.[CrossRef][Web of Science][Medline]

  46. Wilson-Rawls, J., Molkentin, J.D., Black, B.L. and Olson, E.N. (1999) Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C. Mol. Cell. Biol., 19, 2853–2862.[Abstract/Free Full Text]

  47. Tupler, R., Perini, G., Pellegrino, M.A. and Green, M.R. (1999) Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy. Proc. Natl Acad. Sci. USA, 96, 12650–12654.[Abstract/Free Full Text]

  48. McKinsey, T.A., Zhang, C.L., Lu, J. and Olson, E.N. (2000) Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature, 408, 106–111.[CrossRef][Medline]

  49. Sandri, M. and Carraro, U. (1999) Apoptosis of skeletal muscles during development and disease. Int. J. Biochem. Cell Biol., 31, 1373–1390.[CrossRef][Web of Science][Medline]

  50. Coyle, P., Philcox, J.C., Carey, L.C. and Rofe, A.M. (2002) Metallothionein: the multipurpose protein Cell. Mol. Life Sci., 59, 627–647.[CrossRef][Web of Science][Medline]

  51. Cherian, M.G. and Apostolova, M.D. (2002) Nuclear localization of metallothionein during cell proliferation and differentiation. Cell. Mol. Biol., 46, 347–356.

  52. Figlewicz, D.A., Sowden, J.E., Haefele, A., Forrester, J.R., Barrett, K., Kavcic, V. and Tawil, R. (2002) Facioscapulohumeral dystrophy: premature activation of the myogenic program? FSH International Consortium Workshop, 14 October, 2003, Baltimore, MD.

  53. Otten, A.D., Firpo, E.J., Gerber, A.N., Brody, L.L., Roberts, J.M. and Tapscott, S.J. (1997) Inactivation of MyoD-mediated expression of p21 in tumor cell lines. Cell Growth Differ., 8, 1151–1160.[Abstract]

  54. Yip, D.J. and Picketts, D.J. (2003) Increasing D4Z4 repeat copy number compromises C2C12 myoblast differentiation. FEBS Lett., 537, 133–138.[CrossRef][Web of Science][Medline]

  55. Lemmers, R.J.L.F., de Kievit, P., Sandkuijl, L., Padberg, G.W., van Ommen, G.J.B., Frants, R.R. and van der Maarel, S.M. (2002) Facioscapulohumeral muscular dystrophy is uniquely associated with one of the two variants of the 4q subtelomere. Nature Genet., 32, 235–236.[CrossRef][Web of Science][Medline]

  56. Van Overveld, P.G.M., Lemmers, R.J.F., Sandkuijl, L.A., Enthoven, L., Winokur, S.T., Bakels, F., Padberg, G.W., van Ommen, G.-J.B., Frants, R. and van der Maarel, S.M. (2003) D4Z4 hypomethylation in 4q-linked FSHD and non 4q-linked FSHD. Nat. Genet. (in press).

  57. Cremer, T. and Cremer, C. (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet., 2, 292–301.[CrossRef][Web of Science][Medline]

  58. Baur, J.A., Zou, Y., Shay, J.W. and Wright, W.E. (2001) Telomere position effect in human cells. Science, 292, 2075–2077.[Abstract/Free Full Text]

  59. Galy, V., Olivo-Marin, J.C., Scherthan, H., Doye, V., Rascalou, N. and Nehrbass, U. (2000) Nuclear pore complexes in the organization of silent telomeric chromatin. Nature, 403, 108–112.[CrossRef][Medline]

  60. Burke, B. and Stewart, C.L. (2002) Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol., 3, 575–585.[CrossRef][Web of Science][Medline]

  61. Ostlund, C. and Worman, H.J. (2003) Nuclear envelope proteins and neuromuscular diseases. Muscle Nerve, 27, 393–406.[CrossRef][Web of Science][Medline]

  62. Hutchison, C.J. (2002) Lamins: building blocks or regulators of gene expression? Nat. Rev. Mol. Cell. Biol., 3, 848–858.[CrossRef][Web of Science][Medline]

  63. Forrester, W.C., Fernandez, L.A. and Grosschedl, R. (1999) Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer–promoter interactions. Genes Dev., 13, 3003–3014.[Abstract/Free Full Text]

  64. Stratling, W.H. and Yu, F. (1999) Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP. Crit. Rev. Eukaryot. Gene Expr., 9, 311–318.[Web of Science][Medline]

  65. Long, A.D., Mangalam, H.J., Chan, Y., Tolleri, L., Hatfield, G.W. and Baldi, P. (2001) Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12. J. Biol. Chem., 276, 19937–19944.[Abstract/Free Full Text]

  66. Baldi, P. and Hatfield, G.W. (2002) Improved statistical inference from DNA array data using a Bayesian statistical framework. In DNA Microarrays and Gene Expression. Cambridge University Press, Cambridge, pp. 109–119.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
V. Papalouka, D. A. Arvanitis, E. Vafiadaki, M. Mavroidis, S. A. Papadodima, C. A. Spiliopoulou, D. T. Kremastinos, E. G. Kranias, and D. Sanoudou
Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle
Mol. Cell. Biol., November 15, 2009; 29(22): 6046 - 6058.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
X. Xu, K. Tsumagari, J. Sowden, R. Tawil, A. P. Boyle, L. Song, T. S. Furey, G. E. Crawford, and M. Ehrlich
DNaseI hypersensitivity at gene-poor, FSH dystrophy-linked 4q35.2
Nucleic Acids Res., October 9, 2009; (2009) gkp833v1.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
L. Snider, A. Asawachaicharn, A. E. Tyler, L. N. Geng, L. M. Petek, L. Maves, D. G. Miller, R. J.L.F. Lemmers, S. T. Winokur, R. Tawil, et al.
RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: new candidates for the pathophysiology of facioscapulohumeral dystrophy
Hum. Mol. Genet., July 1, 2009; 18(13): 2414 - 2430.
[Abstract] [Full Text] [PDF]

Home page
 DMMHome page
R. D. Wuebbles, M. L. Hanel, and P. L. Jones
FSHD region gene 1 (FRG1) is crucial for angiogenesis linking FRG1 to facioscapulohumeral muscular dystrophy-associated vasculopathy
Dis. Model. Mech., May 1, 2009; 2(5-6): 267 - 274.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
P. Arashiro, I. Eisenberg, A. T. Kho, A. M. P. Cerqueira, M. Canovas, H. C. A. Silva, R. C. M. Pavanello, S. Verjovski-Almeida, L. M. Kunkel, and M. Zatz
Transcriptional regulation differs in affected facioscapulohumeral muscular dystrophy patients compared to asymptomatic related carriers
PNAS, April 14, 2009; 106(15): 6220 - 6225.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
I. Eisenberg, N. Novershtern, Z. Itzhaki, M. Becker-Cohen, M. Sadeh, P. H.G.M. Willems, N. Friedman, W. J.H. Koopman, and S. Mitrani-Rosenbaum
Mitochondrial processes are impaired in hereditary inclusion body myopathy
Hum. Mol. Genet., December 1, 2008; 17(23): 3663 - 3674.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
K. Tsumagari, L. Qi, K. Jackson, C. Shao, M. Lacey, J. Sowden, R. Tawil, V. Vedanarayanan, and M. Ehrlich
Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers
Nucleic Acids Res., April 1, 2008; 36(7): 2196 - 2207.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
K. Hojlund, Z. Yi, H. Hwang, B. Bowen, N. Lefort, C. R. Flynn, P. Langlais, S. T. Weintraub, and L. J. Mandarino
Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS
Mol. Cell. Proteomics, February 1, 2008; 7(2): 257 - 267.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
A. Petrov, J. Allinne, I. Pirozhkova, D. Laoudj, M. Lipinski, and Y. S. Vassetzky
A nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking activity in vivo: Implications for the facio-scapulo-humeral dystrophy
Genome Res., January 1, 2008; 18(1): 39 - 45.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Dixit, E. Ansseau, A. Tassin, S. Winokur, R. Shi, H. Qian, S. Sauvage, C. Matteotti, A. M. van Acker, O. Leo, et al.
DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1
PNAS, November 13, 2007; 104(46): 18157 - 18162.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
I. Eisenberg, A. Eran, I. Nishino, M. Moggio, C. Lamperti, A. A. Amato, H. G. Lidov, P. B. Kang, K. N. North, S. Mitrani-Rosenbaum, et al.
Distinctive patterns of microRNA expression in primary muscular disorders
PNAS, October 23, 2007; 104(43): 17016 - 17021.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
S. A. Greenberg and G. W. Padberg
Pushing the genetic frontier with facioscapulohumeral muscular dystrophy
Neurology, February 20, 2007; 68(8): 544 - 545.
[Full Text] [PDF]

Home page
 NeurologyHome page
R. J. Osborne, S. Welle, S. L. Venance, C. A. Thornton, and R. Tawil
Expression profile of FSHD supports a link between retinal vasculopathy and muscular dystrophy
Neurology, February 20, 2007; 68(8): 569 - 577.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
D. Sanoudou, M. A. Corbett, M. Han, M. Ghoddusi, M.-A. T. Nguyen, N. Vlahovich, E. C. Hardeman, and A. H. Beggs
Skeletal muscle repair in a mouse model of nemaline myopathy
Hum. Mol. Genet., September 1, 2006; 15(17): 2603 - 2612.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Petrov, I. Pirozhkova, G. Carnac, D. Laoudj, M. Lipinski, and Y. S. Vassetzky
Chromatin loop domain organization within the 4q35 locus in facioscapulohumeral dystrophy patients versus normal human myoblasts
PNAS, May 2, 2006; 103(18): 6982 - 6987.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
M. Bakay, Z. Wang, G. Melcon, L. Schiltz, J. Xuan, P. Zhao, V. Sartorelli, J. Seo, E. Pegoraro, C. Angelini, et al.
Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration
Brain, April 1, 2006; 129(4): 996 - 1013.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
T Rijkers, G Deidda, S van Koningsbruggen, M van Geel, R J L F Lemmers, J C T van Deutekom, D Figlewicz, J E Hewitt, G W Padberg, R R Frants, et al.
FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients
J. Med. Genet., November 1, 2004; 41(11): 826 - 836.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
R. Tam, K. P. Smith, and J. B. Lawrence
The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres
J. Cell Biol., October 25, 2004; 167(2): 269 - 279.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
P. S. Masny, U. Bengtsson, S.-A. Chung, J. H. Martin, B. van Engelen, S. M. van der Maarel, and S. T. Winokur
Localization of 4q35.2 to the nuclear periphery: is FSHD a nuclear envelope disease?
Hum. Mol. Genet., September 1, 2004; 13(17): 1857 - 1871.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/22/2895    most recent
ddg327v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (46)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Winokur, S. T.
Right arrow Articles by Flanigan, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winokur, S. T.
Right arrow Articles by Flanigan, K. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?