The role of utrophin in the potential therapy of Duchenne muscular dystrophy

Neuromuscular Disorders 12 (2002) S78–S89 www.elsevier.com/locate/nmd

The role of utrophin in the potential therapy of Duchenne muscular dystrophy Kelly J. Perkins, Kay E. Davies* MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

Abstract Duchenne muscular dystrophy is an X-linked recessive muscle wasting disease caused by the absence of the muscle cytoskeletal protein, dystrophin. Dystrophin is a member of the spectrin superfamily of proteins and is closely related in sequence similarity and functional motifs to three proteins that constitute the dystrophin related protein family, including the autosomal homologue, utrophin. An alternative strategy circumventing many problems associated with somatic gene therapies for Duchenne muscular dystrophy has arisen from the demonstration that utrophin can functionally substitute for dystrophin and its over-expression in muscles of dystrophin-null transgenic mice completely prevents the phenotype arising from dystrophin deficiency. One potential approach to increase utrophin levels in muscle for possible therapeutic purpose in humans is to increase expression of the utrophin gene at a transcriptional level via promoter activation. This has lead to an interest in the identification and manipulation of important regulatory regions and/or molecules that increase the expression of utrophin and their delivery to dystrophin-deficient tissue. As pre-existing cellular mechanisms are utilized, this approach would avoid many problems associated with conventional gene therapies. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Utrophin; Dystrophin; Promoter; Therapeutic strategy; Transcriptional regulation

1. Introduction This review aims to focus on strategies and issues related to up-regulating utrophin expression for DMD therapy. Recent advances of our knowledge of the utrophin gene, in terms of its similarity to dystrophin and its functional capacity to prevent a dystrophic phenotype in dystrophindeficient mdx mice will be discussed. In addition, we will detail recent promoter studies that have uncovered potential pathways of up-regulation and techniques that are in current use to delineate further regulatory regions/elements for this purpose.

2. Structure and protein binding partners of utrophin Utrophin was initially named DMDL (Duchenne muscular dystrophy like) due to its extensive homology to dystrophin, and was originally identified as a 13 kb transcript via a low-stringency screening of a human foetal muscle library using a 3 0 dystrophin probe [1]. The utrophin gene was localized to human chromosome 6q24 [1,2] and the murine equivalent to the proximal region of chromosome 10 [3]. * Corresponding author. Tel.: 144-1865-272179; fax: 144-1865272420. E-mail address: [email protected] (K.E. Davies).

Initial examination of the primary sequence and protein structure of utrophin revealed clear similarities to dystrophin [3]. The N-terminal domain of utrophin, encompassing the first 250 amino acids binds F-actin with similar affinity as dystrophin, although the process is differentially regulated by Ca 21/calmodulin [4–7]. Along with the cysteinerich and C-terminal domains, this region is 80% identical to the equivalent domain of dystrophin [3]. It is therefore not unexpected that utrophin shares similarity in C-terminal protein binding partners [8], commonly called the dystrophin associated protein complex (DAPC) including the dystroglycans [9] and syntrophins [10]. The rod domain of utrophin and dystrophin consists of a number of spectrinlike repeats, with proline-rich hinge regions [3] and is the least conserved region between the two proteins (35% [11]), implying looser evolutionary constraints. The utrophin protein is ubiquitously expressed [12–17], although not uniformly distributed at a subcellular level. In muscle, utrophin is expressed in intramuscular nerves, blood vessels and myofibres. Within the adult myofibre, utrophin is preferentially concentrated in acetylcholine receptor (ACHR)-rich crests at the neuromuscular junction (NMJ), where it binds to components of the DAPC [13,17–20] and myotendinous junctions (MTJ) [13], whereas dystrophin predominates at the sarcolemma. Although the precise role of utrophin in these regions is still unclear, it is thought to

0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S09 60- 8966(02)0008 7-1

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

play an important role in the structure of the post-synaptic cytoskeleton [21]. Given overall domain binding similarities, it has been postulated that utrophin may be the autosomal foetal form of dystrophin and function in a similar manner. This is in part due to the observation that utrophin localizes to the sarcolemma in foetal skeletal muscle from which it disappears and is postnatally substituted by dystrophin [22]. However, despite their structural and functional similarity, the two proteins exhibit distinct localization in normal adult tissues and are expressed in a seemingly reciprocal manner, suggesting co-ordinate regulation. In mature muscle, both are found in the neuromuscular junctions (NMJ) albeit with distinct subjunctional localization. Both are also present in vascular smooth muscles [23] and cardiomyocytes [24] where distinct sub-cellular patterns are displayed. In addition, different isoforms of both proteins have been described. Full-length dystrophin consists of four isoforms driven by different promoters expressed in a tissue-specific pattern [25,26] whereas two utrophin full-length proteins have been described [27–29]. Four (dystrophin) and three (utrophin) short gene products giving rise to C-terminal isoforms have also been described that are expressed in non-muscle tissues. These are transcribed from independent promoters which lie in the introns towards the carboxyterminal region of the gene and have unique 5 0 ends but use the same 3 0 end exons as the full length genes [30–33]. Thus, all these smaller protein products contain the dystrophin/utrophin-protein complex binding domains and are therefore probably linked with the extracellular matrix in non-muscle tissues. The function of these various isoforms is not clear, although we will discuss their corresponding promoters in this review. 3. Utrophin can replace dystrophin in dystrophic muscle After the cloning of the utrophin gene, a number of observations led to the hypothesis that utrophin may functionally compensate for dystrophin deficiency, and perform complementing roles in normal functional or developmental pathways in muscle. As previously mentioned, a high degree of similarity between utrophin and dystrophin exists, especially regarding functionally important protein domains [3,11] and binding partners in muscle [19]. Leading favourably from this observation, in certain myopathies (including DMD), utrophin is re-distributed to the sarcolemma and is often associated with significant increases in utrophin levels. Additionally, utrophin is up-regulated during periods where there is lack of necrosis in dystrophin deficient muscle [13,22,34]. 3.1. Utrophin and dystrophin have complementary functions in muscle Although utrophin is present at high levels developmentally in foetal muscle and satellite cells, levels gradually decline as the expression of dystrophin increases, leading

S79

to its spatially restricted adult expression pattern, although the molecular events giving rise to down-regulation in maturing muscle are not clear. It is hypothesized that dystrophin may be more specialized and adapted to dealing with the much greater stresses of skeletal and cardiac muscle development during birth. Until this occasion, utrophin may give some form of structural rigidity to the developing myotubes [35]. This is supported by observations that utrophin is extrasynaptically localized in recently regenerated muscle fibres which express protein isoforms reminiscent of those produced during myogenic development. In addition, initial muscle necrosis in mdx mice occurs only when high perinatal levels of utrophin decline to levels observed in adult tissue, suggesting a protective role for utrophin in the absence of dystrophin [13]. Such delayed onset of DMD parallels observations in which high levels of foetal haemoglobin (HbF) at birth can compensate for defective adult Hb for a restricted time after birth in b-thalassaemia patients, delaying the onset of symptoms until foetal levels decline to subcompensatory levels [36]. Importantly, increased levels of HbF can ameliorate the clinical course of inherited disorders of b-globin gene expression, such as b-thalassaemia and sickle cell anaemia [37]. The mild phenotypic changes seen in mdx mice and utrophin null mutant mice (in the latter, the only pathological change occurs at the neuromuscular junction, in a reduction in the number of AChRs and postsynaptic folding, but with little electrophysiological difference [38]) could conceivably arise from functional redundancy allowing each to compensate for absence of the other. In support of this, utrophin null-mdx (dko) mice have a very severe myopathic phenotype that is lethal within weeks of birth. These mice show many signs typical of DMD in humans, such as progressive muscle weakness, contractures and kyphoscoliosis [38,39]. 3.2. Over-expression of utrophin rescues the mdx phenotype Evidence illustrating that utrophin could functionally compensate for dystrophin deficiency in muscle was initially addressed in 1996 with the creation of a transgenic mouse line expressing a truncated utrophin transgene under the control of a constitutive muscle promoter, bred on to the mdx background [35]. Despite a continuing absence of dystrophin, a dramatic phenotypic improvement was observed in the resulting offspring, with histochemical analysis showing diminutive evidence of the fibrosis and necrosis normally observed, especially in the diaphragm (the most severely affected organ in the mouse model). On a cellular level, a reduction in the proportion of myofibres in limb and diaphragm muscles with central nuclei was observed, indicating a reduction in the amount of muscle regeneration compared with control mdx mice. In addition, serum creatine kinase (a marker of sarcolemmal permeability and cell damage) was reduced to near control levels. Immunohistology showed that the truncated utrophin

S80

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

protein had localized to the sarcolemma, where components of the DAPC had been restored [35]. Recent studies have shown that the overexpression of full-length recombinant utrophin can rescue the defective linkage between costameric actin filaments and the sarcolemma in mdx muscle [40], indicating that utrophin and dystrophin are functionally interchangeable actin binding proteins. This recent evidence is supported by the improvement of several physiological parameters in the original transgenic study, including the mean normalized tetanic force, force drop after sarcolemmal disruption, eccentric contraction and Ca 21 homeostasis [41] and in a 1998 study of mdx mice expressing a full-length transcript [42]. Both of the transgenic studies signified that the utrophin levels required in muscle were significantly less than the normal endogenous utrophin levels seen in lung and kidney, and that pathology depended on the amount of utrophin expression. The highest expressing lines (approximately 10-fold higher than endogenous levels) were able to affect almost complete phenotypic rescue when bred onto the mdx background, both in morphological and physiological tests [42]. Quantitative analysis indicated that morphological and functional recovery was achieved with levels of muscle protein expression that were 2- to 3-fold higher than wild-type muscle. This level of expression was about 50% of the normal wild-type level found in the kidney and approximately 25% of the endogenous level in the lung. More recently, importantly, high-level ubiquitous expression of utrophin had no resultant toxicity [35,38,42,43].

4. Dystrophin and utrophin promoters Dystrophin mRNA expression is one of the most highly regulated in the cytoskeletal protein family [30,44,45], as transcription occurs from several independently regulated promoters, allowing regulation on a developmental and tissue-dependent level. Of these, the cortical (C), muscle (M), and Purkinje (P) promoters express full-length 14 kb transcripts consisting of unique first exons that splice into a shared exon 2, with 78 common exons (promoter names indicate major, but not exclusive, sites of expression [46– 49]). Four additional promoters located in introns 29, 55, 59, and 68 encode shorter isoforms Dp 260 [50], Dp 140 [51], Dp 116 [52], and Dp 71 [53], which are predominantly expressed in retina, the central nervous system, the peripheral nervous system, and non-muscle tissues, respectively. The utrophin gene spans approximately 1Mb of genomic DNA and gives rise to a 13 kb transcript [11]. Although ubiquitously expressed, like dystrophin it is highly regulated at the level of developmental and subcellular distribution, and is present in myofibres in a temporal and spatial manner parallel to the AChR [14,54]. Relatively abundant levels persist in adult lung and higher levels in foetal muscle compared to adult skeletal muscle have been observed [15,55]. Utrophin transcripts are also specifically localized during develop-

ment, with initial accumulation in the neural tube and later becoming abundant at a variety of other sites such as the tendon primordia in the digits, the pituitary, thyroid and adrenal glands, cardiac muscle, and the kidney and lung [56]. As similarities exist between both the utrophin and dystrophin genes, it is likely that they were derived as a result of common ancestral gene duplication. Although the precise event giving rise to evolutionary divergence into two loci is unknown, studies have suggested it is likely to have occurred immediately prior to vertebrate radiation and that utrophin is more highly related to dystrophin in comparison to other related proteins, such as DRP-2, located on the X chromosome [57]. Inter-specific divergences for utrophin, however, are approximately 4-fold higher than those observed for dystrophin, suggesting less stringent functional constraints on sequence variation. Although the two identified promoters that give rise to full-length utrophin isoforms (discussed in the next section) do not appear to be conserved in function or genomic location, intronic promoters isolated within the utrophin locus have significant homology to those identified in dystrophin. The first identified was called Gutrophin (or Up116), a 5.5 kb transcript located in sensory ganglia and brain [58,59] corresponding to Dp116 (specific to peripheral nerve [58]). Two alternate intronic transcripts of utrophin have been described; Up71 and Up140 [33], with unique first exons and promoters located in intron 62 and intron 44, respectively. These transcripts are widely expressed in both human and mouse tissues, including skeletal muscle and appear to be structural homologues of the short dystrophin transcripts, Dp140 and Dp71, emphasizing the high degree of structural conservation between the utrophin and dystrophin genes. The expression of short isoforms from the utrophin and dystrophin loci respectively, and the localization of their promoters in similar intronic positions relative to exons (see Fig. 1), strongly suggests ancient duplication. Analysis of the genomic structure of utrophin and dystrophin supports this theory, as both genes contain multiple short exons, a single long exon encoding the 3 0 -untranslated region (UTR) and large intronic regions separating the first and second coding exons.

5. Promoters giving rise to full-length utrophin isoforms Utrophin is unique from dystrophin in the respect that the first exon identified was non-coding [28,29], and is separated from the second exon by a short genomic interval. This transcript was subsequently called ‘A’ as a second independently regulated full-length isoform has been isolated (utrophin B; [27]), giving rise to transcripts with unique 5 0 exons that splice into a common mRNA at exon 3 (Fig. 1). Promoter A lies within a methylation-sensitive CpG island at the 5 0 end of the gene; promoter B lies within the large second exon of utrophin. Current knowledge of the differential expression profiles of the two resulting isoforms indicate that A and B are co-expressed, although independently

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

S81

Fig. 1. Identified human dystrophin and utrophin isoforms and associated promoters. Schematic organization of known promoters, exons and enhancers of dystrophin and utrophin. The exons (not to scale) are represented as follows: dark grey, translated; light grey, untranslated; black, common exons relative to full-length isoforms. Arrows indicate transcription start sites; white regions (with captions) are characterized enhancers. The 5 0 regions of both genomic regions between exons and enhancers to exon 2 for dystrophin and 3 for utrophin are to scale, all other regions are not to scale.

regulated, in a number of tissues. This section will focus on our current knowledge of the structure of the two promoters and transcriptional processes that confer and control expression, including cis-acting enhancer regions. 5.1. The utrophin A promoter Evidence from the specific localization of utrophin at the NMJ indicated that at least one promoter regulates transcription in a manner similar to synaptically expressed genes such as the acetylcholinesterase promoter, which is CGrich, regulated during muscle cell differentiation and localized specifically at the NMJ [60,61]. The genomic region surrounding the first identified utrophin promoter (A) showed striking similarities; the first exon is untranslated, transcribed from multiple start sites, lies within a CpG island [11] and shows an absence of TATA and CCAT motifs common to eukaryotic promoters [29]. The minimal promoter element was identified (155 bp), with the first exon and 900 bp upstream displaying 66% sequence conservation between human and mouse (Fig. 2). Further definition of the CG-rich core element revealed conserved cognate sequences for transcription factors such as Ap2, Sp1 and Sp3 [29], of which Ap2 and Sp1 have been implicated in optimal basal activity [62]. The importance of these ubiqui-

tously expressed factors in conferring a specific expression profile will be further discussed. Two important motifs were isolated within a 1 kb region flanking the core promoter element. A consensus E-box is conserved between both species, defined by the nucleotides CANNTG and is a helix–loop–helix factor-binding site involved in regulating muscle gene expression [63]. In addition, the human and mouse utrophin 5 0 flanking region contain the core sequence of the N-box, an element shown to direct synapse-specific expression of the mouse acetylcholine receptor d-subunit gene [64]. This TTCCGG motif restricts the expression of the d-subunit gene to the NMJ by enhancing expression at the endplate and though hypothetically acting as a silencer in extrajunctional areas. Sequences identical to this core sequence of the N-box are present in other AChR subunits and regulate the synaptic expression of at least some of these genes [64]. The Nbox is located at differing sites in the human and mouse utrophin sequence which is consistent with previous observations that the N-box is not necessarily conserved between the same AChR subunit gene in different species nor between different subunit genes of the same species [64]. The presence of both N- and E-motifs indicate that expression of the A transcript is subject to regulatory mechanisms similar to those previously described for

S82

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

Fig. 2. Sequence conservation between human and mouse at the utrophin A and B loci. Dotplot comparison of the utrophin mouse (horizontal axis) against human (vertical axis) genomic region surrounding exons 1A and 1B. White squares represent the appropriate transcribed exon. The dotplot was assembled using the GCG package, with human sequences numbered according to their position on the PAC ALO24474 (provided through the Sanger Centre Human Chromosome 6 Project Website (http://www.sanger.ac.uk)). The diagonal line indicates the overall degree of homology between the two sequences.

dystrophin [30,65–67] and other synaptic proteins such as the AChR [68–74] during myogenic differentiation. Current knowledge of N- and E-box mediated mechanisms is summarized below (and shown schematically in Fig. 3). 5.2. Synaptic regulation via the N-box Synaptic expression of utrophin in adult muscle is partially attributable to enhanced transcription in sub-synap-

tic nuclei with consequent innervation-associated synaptic accumulation of mRNA. This is similar to the nicotinic acetylcholine receptor d and 1 subunit and acetylcholine esterase genes [75,76] that code for components of the neuromuscular junction. The synapse-specific transcriptional properties of these promoters are conferred via the N-box, therefore the properties of the N-box motif present in the utrophin 5 0 -UTR were investigated. In common with other synaptically expressed muscle

Fig. 3. Positive transcriptional interactions identified in the human A utrophin promoter. Current knowledge on transcriptional activation of the utrophin A isoform. The diagram indicates the A promoter in two interactive sections; the upstream region containing the N- and E-boxes, and the core promoter element. For the core element: Ap2, white boxes; Sp1, light grey. For the Sp1 sites, core promoter activity has been shown with Sp1, whereas Sp3 binds identical sites and is also implicated in interacting with GABP at the N-box. (Note: the binding site(s) of the Sp1/3 factors that associate with GABP are not confirmed.) Multiple transcription start sites are asterisked. For the upstream region, characterized factors that bind the N- and E-boxes and confer transcriptional activation (plus symbol) are as indicated, as is the approximate position of the murine N-box relative to the conserved E-box (N-box m). Distances between motifs are to scale.

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

genes, comprehensive studies show that utrophin experiences an N-box mediated transcriptional response to heregulin and specifically GABP in vitro [77,78]. Initially, a reporter gene driven by a 1.3 kb promoter A fragment (containing the N-box) was preferentially expressed at post-synaptic nuclei in adult muscle [77]. Transcriptional activity by heregulin was abolished through N-box mutagenesis; over-expression of heregulin or GABP (a or b) in mouse and human cultured myotubes caused a N-box dependent increase in promoter activity with a resultant 2.5-fold increase in utrophin mRNA levels, and proteinDNA assays performed with muscle extracts directly implicated GABP binding at the N-box. These findings were confirmed in vivo through direct gene transfer. The signalling cascade used by such factors to stimulate N-box dependent synaptic expression of genes such as utrophin are complex, but are initially thought to occur with these factors stimulating Erk and Jnk MAP kinases, allowing phosphorylation of the ets-related GABP a/b class of transcription factors. This modification is thought to either aid the interaction of GABP with the overlapping utrophin N-box/ets binding site [79] or influence its ability to modify transcription [80]. Subsynaptic stimulation via this signalling pathway appear conserved among genes expressed at the neuromuscular junction [64,75,76,81,82]. Importantly, interactions at the N-box are also modulated by interaction with elements bound to the core promoter element. For example, Sp1 and Sp3, zinc finger containing transcription factors often cooperate with GABP a/b [83,84] and interact with the N-box to stimulate the utrophin promoter [85]. Synergistic activation between the Sp and ets factors in muscle cells may be critical for the regulation of the utrophin promoter [85]. As heregulin and GABP a/b are able to confer transcriptional activation of the utrophin A isoform in cultured muscle cells, it has been suggested that these factors may be employed in the in vivo upregulation of endogenous A transcript in dystrophin deficient muscle [77,79]. In addition, in terms of current knowledge of interactions at the N-box, methods to de-repress the promoter or block the activity of repressor molecules such as the ets domain protein ERF (or ERF-like molecules) and/or trans-activating positively regulatory members of the ets family [86] may be a functional means in which to activate the utrophin A promoter [79]. 5.3. Involvement of the E-box in myogenic induction In contrast to synaptic regulation, until recently, little information was available on the induction of utrophin expression observed during muscle differentiation. This was despite initial observations that the A promoter contains a conserved E-box, a binding site for helix–loop–helix proteins of the MyoD family, including MyoD1 [87], myogenin [88,89], myf5 [90] and MRF4 [91]. E-box motifs are found in the promoters of many muscle-specific genes, and enhance the in vitro transcriptional activity of the a, b

S83

and g AChR subunit genes [73,92,93]. Given the co-localization of utrophin with AChRs at the NMJ, it was therefore of interest as to whether myogenic factors regulate the expression of utrophin. Evidence of muscle-specific transcriptional regulation via the E-box was initially outlined in 1999 with the observation that the rate of total utrophin transcription increased approximately 2-fold during myogenesis in the mouse C2C12 muscle cell line [94]. This is attributed to an increase in endogenous A-utrophin mRNA levels [62] that is paralleled by expression of a construct containing the conserved upstream muscle-specific E-box. The binding of MyoD, myogenin and MRF4 transactivates the A promoter up to 18-fold in myotubes in transient assays [62] and is abolished by mutagenesis of the E-box, confirming that promoter A is regulated in a similar manner to other genes expressed in muscle [61,95–97]. Mechanistically, this type of positive regulation may involve direct interactions between myogenic factors and basal factors bound to the core promoter (Fig. 3), analogous to the association of Sp1 and MyoD-myogenin factors required for activation of the human cardiac a-actin (HCA) promoter in skeletal muscle cells [95]. Using a similar approach postulated for the N-box, E-box mediated activation may be achieved through blocking the activity of potent repressor molecules such as Dermo-1, a multifunctional bHLH (basic helix–loop–helix) transcription factor that inhibits MyoD trans-activation of E-box dependent gene expression [98]. Alternatively, it may be possible to trans-activate utrophin promoter A in DMD muscle by delivery of pharmacological compounds that directly or indirectly alter myogenic factor levels and/or activity. In combination with the N-box, studies on the Ebox underline the importance of these motifs and their interaction with elements in the core promoter in the developmental regulation of the utrophin gene in skeletal muscle. 5.4. The utrophin B promoter In 1999, a second promoter (designated ‘B’) giving rise to a full-length isoform with a unique 31 amino acid first exon (1B) was identified within the large second intron of the human and mouse utrophin genes, 52.2 and approximately 50 kb 3 0 to exon 2, respectively [27]. The human and mouse sequences show 82% nucleotide and 77% translational identity [27]. Sequence analysis indicated that the promoter was of the TATA-Inr 1 type, due to the absence of TATA or CAAT motifs, although unlike promoter A, transcription occurs from a single initiator site, approximately 100 bp from the translation start site. The presence of a short open reading frame within the 5 0 untranslated region prior to the actual translation start is similar to the structure of exon 2A [27–29]. Analysis of the 1.5 kb genomic region 5 0 of human exon 1B conferred orientation-specific activation in reporter analysis and mimicked the transcription profile of the endogenous transcript in three different cell lines, IN157 (rhab-

S84

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

domyosarcoma), CL11T47 (kidney epithelial), and HeLa (cervical epithelial) [27]. It is therefore likely that this region contains both the necessary signals to initiate transcription of exon 1B and regulatory elements that determine the level of expression in these cell lines. A 300 bp element with 74% sequence identity contained within this region was defined as the minimal promoter region, as a 5 0 and 3 0 deletion series analysis retains 70% activity of the full 1.5 kb construct in expressing cell lines [27]. A human and mouse alignment of a 2 kb region of the B locus shows limited overall homology (48%), with the only significant conserved region limited to exon 1B and the core promoter region (Fig. 2). The human B promoter gives rise to a widely expressed transcript, and is most abundant in heart and pancreas relative to b-actin [27]. The utrophin A and B transcripts are coexpressed, albeit independently regulated, for example, B transcripts are more abundant in heart, whereas exon 2Acontaining transcripts predominate in the kidney [27–29]. It is conceivable, however, that examining transcript distribution levels in whole tissue has masked the possibility of cell type-specific patterns of expression; this is strongly supported by the very high variation of promoter activity ( < 10-fold) within cell lines in vitro. The chance also exists that the two promoters may be spatially regulated in a subcellular manner. Available data therefore indicates that B is independently regulated from A and responds to a discrete group of cellular signals. In contrast to extensive studies undertaken with the A promoter, and the potential for up-regulation of B as an alternate therapeutic target (and thus increasing small compound screening yields), it is somewhat surprising that there are no published data to date of the transcriptional mechanisms governing its expression. This may, in part, be due to the lack of noticeable motifs that confer specific expression, such as N- and E-boxes. However, understanding of transcriptional processes at the B locus is desirable as a promoter without synaptic regulatory elements may circumvent interference with mechanisms regulating expression of other postsynaptic components that may lead to impairment of the structure and function of the NMJ. It is also possible that up-regulation of utrophin B may be necessary to ameliorate the dystrophic phenotype. In adult skeletal muscle fibres, promoter A is synaptically driven [54,78], yet aggregates of utrophin mRNA are detectable at up to 25% of extrasynaptic nuclei, of which expression of the B isoform may be invoked as one possible explanation. In addition, as cardiac dysfunction is a common feature of dystrophinopathies [99], manipulation of utrophin A alone may not be therapeutically sufficient if the cardiac utrophin message was transcribed from an alternate promoter. In order to address these questions, we have instigated a number of approaches in our laboratory. Similar to our studies on utrophin A [62], we are investigating the Bpromoter region in order to delineate mechanisms and factors that confer the large cellular differences in transcrip-

tion observable in vitro. On a protein level, we are investigating the subcellular distribution patterns of utrophin A and B proteins using recently developed isoform-specific antibodies. The latter will assist in a more focused search for specific elements/motifs in the promoter region that may confer localized transcription within regions where the protein is detected.

6. Alternate modulators of promoter activity Given the genomic similarity with dystrophin and the complexity of utrophin expression, there is a distinct possibility that additional utrophin promoters are present. Transcription from either identified or undisclosed promoters may rely on supplementary cis-acting regulators. In the dystrophin locus, a cis-acting enhancer, located 6.5 and 8.5 kb downstream from the muscle (M) promoter in the human and mouse sequences respectively influence promoter activity [47,100]. The sequence is 65% conserved and is approximately 200 bp in length for both species. The muscle-specific human dystrophin muscle enhancer (DME1; [47]) increases M promoter activity in immature and mature skeletal muscle and contains three MEF-1/Ebox and two MEF-2/AT-rich motifs as potential musclespecific regulatory domains [100]. Interestingly, this region has been implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy [101]. The mouse enhancer contains three functional E-boxes (of which two bind MyoD) and a serum response element; all are essential for enhancer activity in myotubes, although the latter is implicated in transcriptional repression in myoblasts [100]. The corresponding region in the utrophin gene was studied using a reporter system of genomic fragments, and a new regulatory element in the human sequence called the downstream utrophin enhancer (DUE) was identified [102]. The element mapped downstream (within the second intron) of the utrophin A transcription start site, co-localized with a DNase I-hypersensitive site and was localized to a 128 bp region that retained complete activity. The element behaves as a classic enhancer as it is orientation-independent and enhances both the utrophin A and heterologous thymidine kinase promoters. It is of interest that regulatory elements localized in the utrophin enhancer do not confer increased activity during myogenic differentiation [102], suggesting that processes occurring via the E-box in the A promoter do not require DUE localized regulatory elements. Consistent with the ability of the enhancer to confer activity in nonmuscle cell lines, no muscle-specific elements were identified, although consensus sequences to the transcription factor families Ap1 and GATA were located. Given that the dystrophin enhancer is conserved between mouse and humans, it would be of interest to identify a similar acting element in the mouse sequence, and whether activation is specific to the A promoter or influences additional promoters.

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

This section has concentrated on studies identifying or characterizing mechanisms that regulate the transcription of utrophin. These studies are essential in order to understand utrophin regulation in muscle cells and represent a first step forward for drug design with the aim of an utrophin based therapeutic strategy for DMD, as discussed below.

7. Utrophin-based therapeutic strategies An observable degree of functional redundancy between dystrophin and utrophin indicates that their distinct functions relate more to discrete expression patterns rather than differences in biochemical or physical properties. This has been functionally demonstrated by the ability of utrophin over-expression to rescue the dystrophic phenotype in mdx muscle, and allows consideration of possible therapeutic strategies. The first addresses the direct delivery of utrophin protein to muscle. This approach may avoid the potential problems of an immune response associated with dystrophin delivery in patients and is consequently a more preferable option. Favourably, recent evidence has suggested that expression of an utrophin transgene is more prolonged than dystrophin following adenoviral delivery to the muscles of immune competent mice [103]. Successful adenoviral delivery of utrophin to muscle has been demonstrated, with phenotypic improvement both in mdx [104] and dko muscle [105]. In the latter study, delivery of a first generation recombinant adenovirus containing an utrophin minigene to the limb muscle (tibialis anterior) of dko neonatal mice protected the muscle from subsequent dystrophic damage [105]. Expression of the minigene was detectable in up to 95% of fibres 30 days post injection and caused a significant decrease in necrosis. Importantly, these observations show that introducing the utrophin transgene after the onset of muscle necrosis and regeneration can correct the dystrophic phenotype. Although studies mentioned in this review have been crucial in demonstrating the protective role of utrophin overexpression in transgenic mdx and dko mice (either in utero or via viral somatic transfer), several important insights are required for eventual therapeutic use. For example, it is necessary to understand whether expression of utrophin in muscle cells is equally effective at early and later stages of disease progression and if large quantities of utrophin in muscle results in short or long term benefits. An avenue of current research in our laboratory that begins to address such uncertainties is the use of tetracyclineresponsive transactivator (tTA) analysis [106]. This system allows the transcription of any gene to be somatically induced (or repressed) in multiple muscle groups at any point throughout the life of the mouse by the administration of tetracycline, and has been successful in determining the timing of controlled induction of dystrophin in the mdx mouse in order to prevent dystrophic pathology [107]. In this study, expression of dystrophin in utero was found to

S85

result in a more dramatic improvement in muscle morphology than induction within a few days after birth. Importantly, induced dystrophin expression after 4 weeks of age did not result in obvious phenotypic improvement or positive morphological change in muscle. We are currently undertaking similar studies for utrophin in the mdx mouse using the tTA system to determine the developmental period in which utrophin delivery is most effective in preventing the dystrophic phenotype. The second therapeutic possibility involves the prevention of down-regulation and/or up-regulation of the endogenous utrophin gene in skeletal muscle sufficient to effect the 2- to 3-fold increase in steady-state protein levels necessary to prevent dystrophic pathology. This may be achieved through the use of small diffusible chemical compounds and has an inherent advantage of circumventing the challenge of conferring stable expression of transgenes in skeletal muscle. Such an approach has been successful in the treatment of b-thalassaemia, which results from mutations in the b-globin gene. The foetal isoform, g-globin is down regulated after birth, such that a switch between foetal (a2g2) and adult (a2b2) forms of haemoglobin occurs by 3–6 months of age (reviewed in Ref. [108]). Reactivation of transcription from the g-globin locus in red cell precursors can functionally compensate for b-globin deficiency [109]. Small compound treatment with butyrate derivatives are able to effect such reactivation through interaction with 5 0 regulatory elements of the g-globin promoter [110–112]. In some instances, changes in the patterns of DNA–protein interactions have been demonstrated in red cell precursors of patients pre and post treatment [113]. Early clinical trials have shown several beneficial, albeit variable effects [109,114,115] and illustrate an important principle with respect to validating a similar approach for utrophin. Patients have improved clinically following small compound transcriptional manipulation of genes encoding functionally similar proteins, achieving compensation for the absence of one protein by effecting up-regulation of another. Importantly, pharmacological compounds do not necessarily require strict tissue-specific control and is aided by the observation that ubiquitous over-expression of the target protein has no resulting toxicity in the mdx mouse [35,41,43,116]. The identification and characterization of transcriptional mechanisms within the utrophin promoters allows an opportunity to specifically design small compounds that interact with or target these processes [117]. A systematic approach of investigating protein-DNA/protein-protein interactions of the proximal promoter regions and the use of in vitro reporter analysis have been successful in the recognition of a number of potential pathways for regulating expression via the N- and E-boxes of the utrophin A promoter. Further understanding of trans-acting factors and appropriate signalling pathways at these elements may assist in the design of specific molecules (such as heregulin) to target specific events to yield transcriptional activation. For exam-

S86

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

ple, current knowledge of transcriptional interactions at the N-box has led to the recent evaluation of l-arginine as a utrophin-targeting compound for possible therapeutic use. This molecule is a limiting substrate for nitric oxide (NO) biosynthesis, which hypothetically mediates a signalling pathway that regulates agrin-induced aggregation of synapse-specific components at the NMJ, including utrophin [118]. Studies of adult normal and mdx mice (and in corresponding myoblast lines in vitro) indicated that treatment with l-arginine, NO, or hydroxyurea (an intermediate compound in the l-arginine–NO pathway) increased utrophin levels and enhanced sarcolemmal localization [119,120]. However, we are unable to recapitulate these findings using identical conditions, finding no such effect on utrophin transcription and/or protein levels upon delivery of either l-arginine or hydroxyurea in vitro (unpublished observations). An alternate approach to identifying suitable molecules that increase utrophin levels is the screening of random small compound libraries against an easily quantifiable automated assay. As an example, transcriptional up-regulation of either utrophin full-length isoform may be affected through the use of reporter gene-cell culture systems (described in Ref. [28]). We are currently using this system in our laboratory as it has the inherent advantage of being able to identify additional compounds that positively interact via unknown mechanisms. A larger scale concurrent approach is also under way to isolate and clone elements that modulate promoter activity such as cis-acting elements, used for the recent identification of the utrophin intronic enhancer [102]. A strategy that allows the analysis of greater genomic regions ð.140 kb) and circumvents limitations of plasmid-based approaches involves BAC transgenesis. This homologous-recombination based method has been successful in the study of the myogenic genes myf-5 and MRF4 [121] where the resultant transgenic mice reproduced all known aspects of temporal and spatial expression from both loci. Importantly, novel elements were identified for myf-5 that were responsible for specific expression in individual cell populations and enabled the localization of multiple elements required for recapitulating the endogenous expression pattern of MRF4. The BAC approach would therefore be extremely useful for (1) analysing expression patterns of A and B utrophin isoforms within an identical genomic and developmental context and (2) delineating novel regulatory elements residing within the BAC. The availability of utrophin A and B antibodies will assist in confirming in vivo expression of the reporter proteins from the utrophin locus and provide an additional resource though which regions of transcriptional and protein distribution can be compared. Given the ability of utrophin to serve as a functional replacement for the absence of dystrophin, this review has concentrated on studies of the molecular physiology of utrophin expression in order to identify the various mechanisms that give rise to the complex expression pattern in healthy

and diseased tissue. Current knowledge on its transcriptional processes as a resource to enable identification of means to effect its up-regulation in dystrophic tissue lends support to the continued characterization of utrophin as a worthwhile avenue of research for a definitive cure for Duchenne muscular dystrophy. Acknowledgements We gratefully acknowledge Allyson Potter and Jeff Ehmsen for discussions and manuscript suggestions, and Dr Andrew Weir for assistance with Fig. 2. K.J.P. is a current recipient of the Sir Edward Dunlop Memorial Award (Aust.) and acknowledges previous support from the ORS scheme. This work is supported the Medical Research Council (UK), the Muscular Dystrophy Campaign (UK), the Muscular Dystrophy Associations of USA and South Australia, and the Association Francaise Contre les Myopathies. References [1] Love DR, Hill DF, Dickson G, et al. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature 1989;339:55–58. [2] Buckle VJ, Guenet JL, Simon Chazottes D, Love DR, Davies KE. Localisation of a dystrophin-related autosomal gene to 6q24 in man, to mouse chromosome 10 in the region of the dystrophia muscularis (dy) locus. Hum Genet 1990;85:324–326. [3] Tinsley JM, Blake DJ, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992;360:591–593. [4] Morris GE, Nguyen TM, Nguyen TN, Pereboev A, Kendrick Jones J, Winder SJ. Disruption of the utrophin-actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin. Biochem J 1999;337:119–123. [5] Winder SJ, Hemmings L, Bolton SJ, et al. Calmodulin regulation of utrophin actin binding. Biochem Soc Trans 1995;23:397s. [6] Winder SJ, Hemmings L, Maciver SK, et al. Utrophin actin binding domain: analysis of actin binding and cellular targeting. J Cell Sci 1995;108:63–71. [7] Winder SJ, Kendrick Jones J. Calcium/calmodulin-dependent regulation of the NH2-terminal F-actin binding domain of utrophin. FEBS Lett 1995;357:125–128. [8] James M, Simmons C, Wise CJ, Jones GE, Morris GE. Evidence for a utrophin-glycoprotein complex in cultured cell lines and a possible role in cell adhesion. Biochem Soc Trans 1995;23:398s. [9] James M, Nguyen TM, Wise CJ, Jones GE, Morris GE. Utrophindystroglycan complex in membranes of adherent cultured cells. Cell Motil Cytoskeleton 1996;33:163–174. [10] Peters MF, Adams ME, Froehner SC. Differential association of syntrophin pairs with the dystrophin complex. J Cell Biol 1997;138:81–93. [11] Pearce M, Blake DJ, Tinsley JM, Byth BC, Campbell L, Monaco AP, Davies KE. The utrophin and dystrophin genes share similarities in genomic structure. Hum Mol Genet 1993;2:1765–1772. [12] Blake DJ, Tinsley JM, Davies KE. Utrophin: a structural and functional comparison to dystrophin. Brain Pathol 1996;6:37–47. [13] Khurana TS, Watkins SC, Chafey P, et al. Immunolocalisation and developmental expression of dystrophin related protein in skeletal muscle. Neuromuscul Disord 1991;1:185–194. [14] Khurana TS, Watkins SC, Kunkel LM. The subcellular distribution

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26] [27]

[28] [29]

[30] [31] [32]

[33]

[34]

[35]

of chromosome 6-encoded dystrophin-related protein in the brain. J Cell Biol 1992;119:357–366. Love DR, Morris GE, Ellis JM, et al. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse. Proc Natl Acad Sci USA 1991;88:3243–3247. Nguyen TM, Ellis JM, Love DR, et al. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J Cell Biol 1991;115:1695–1700. Nguyen TM, Le TT, Blake DJ, Davies KE, Morris GE. Utrophin, the autosomal homologue of dystrophin, is widely-expressed and membrane-associated in cultured cell lines. FEBS Lett 1992;313:19–22. Campanelli JT, Roberds SL, Campbell KP, Scheller RH. A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering. Cell 1994;77:663–674. Matsumura K, Ervasti JM, Ohlendieck K, Kahl SD, Campbell KP. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 1992;360:588–591. Ohlendieck K, Ervasti JM, Matsumura K, Kahl SD, Leveille CJ, Campbell KP. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 1991;7:499–508. Love DR, Byth BC, Tinsley JM, Blake DJ, Davies KE. Dystrophin and dystrophin-related proteins: a review of protein and RNA studies. Neuromuscul Disord 1993;3:5–21. Clerk A, Morris GE, Dubowitz V, Davies KE, Sewry CA. Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle. Histochem J 1993;25:554–561. Rivier F, Robert A, Hugon G, Mornet D. Different utrophin and dystrophin properties related to their vascular smooth muscle distributions. FEBS Lett 1997;408:94–98. Pons F, Robert A, Fabbrizio E, et al. Utrophin localization in normal and dystrophin-deficient heart. Circulation 1994;90:369–374. Sadoulet Puccio HM, Rajala M, Kunkel LM. Dystrobrevin and dystrophin: an interaction through coiled-coil motifs. Proc Natl Acad Sci USA 1997;94:12413–12418. Winder SJ. The membrane-cytoskeleton interface: the role of dystrophin and utrophin. J Muscle Res Cell Motil 1997;18:617–629. Burton EA, Tinsley JM, Holzfeind PJ, Rodrigues NR, Davies KE, second A. promoter provides an alternative target for therapeutic upregulation of utrophin in Duchenne muscular dystrophy. Proc Natl Acad Sci USA 1999;96:14025–14030. Dennis CL. Promoter studies of the utrophin gene. Doctor of Philosophy thesis. Oxford: University of Oxford, 1996. Dennis CL, Tinsley JM, Deconinck AE, Davies KE. Molecular and functional analysis of the utrophin promoter. Nucleic Acids Res 1996;24:1646–1652. Ahn AH, Kunkel LM. The structural and functional diversity of dystrophin. Nat Genet 1993;3:283–291. Ahn AH, Kunkel LM. Syntrophin binds to an alternatively spliced exon of dystrophin. J Cell Biol 1995;128:363–371. Lederfein D, Yaffe D, Nudel U. A housekeeping type promoter, located in the 3 0 region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene. Hum Mol Genet 1993;2:1883–1888. Wilson J, Putt W, Jimenez C, Edwards Y. Up71 and Up140, two novel transcripts of utrophin that are homologues of short forms of dystrophin. Hum Mol Genet 1999;8:1271–1278. Karpati G, Carpenter S, Morris GE, Davies KE, Guerin C, Holland P. Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle. J Neuropathol Exp Neurol 1993;52:119–128. Tinsley JM, Potter AC, Phelps SR, Fisher R, Trickett JI, Davies KE. Amelioration of the dystrophic phenotype of mdx mice using a

[36]

[37] [38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52] [53]

[54]

[55]

[56]

S87

truncated utrophin transgene [see comments]. Nature 1996;384:349–353. Vrettou C, Kanavakis E, Traeger-Synodinos J, et al. Molecular studies of beta-thalassemia heterozygotes with raised Hb F levels. Hemoglobin 2000;24:203–220. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci 1998;850:38–44. Deconinck AE, Rafael JA, Skinner JA, et al. Utrophin-dystrophindeficient mice as a model for Duchenne muscular dystrophy. Cell 1997;90:717–727. Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS, Sanes JR. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 1997;90:729–738. Rybakova IN, Patel JR, Davies KE, Yurhcenco PD, Ervasti JM. Utrophin binds laterally along actin filaments and can couple costameric actin with the sarcolemma when overexpressed in dystrophindeficient muscle. Mol Biol Cell 2002;13:1512–1521. Deconinck N, Tinsley J, De Backer F, et al. Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice. Nat Med 1997;3:1216–1221. Tinsley J, Deconinck N, Fisher R, Kahn D, Phelps S, Gillis JM, Davies K. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med 1998;4:1441–1444. Fisher R, Tinsley JM, Phelps SR, et al. Non-toxic ubiquitous overexpression of utrophin in the mdx mouse. Neuromuscul Disord 2001;11:713–721. Sironi M, Bardoni A, Felisari G, et al. Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle. J Neurol Sci 2001;186:51–57. Sironi M, Pozzoli U, Cagliani R, Comi GP, Bardoni A, Bresolin N. Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms. Hum Genet 2001;109:73–84. Feener CA, Koenig M, Kunkel LM. Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature 1989;338:509–511. Klamut HJ, Bosnoyan Collins LO, Worton RG, Ray PN, Davis HL. Identification of a transcriptional enhancer within muscle intron 1 of the human dystrophin gene. Hum Mol Genet 1996;5:1599–1606. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50:509–517. Nudel U, Zuk D, Einat P, et al. Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature 1989;337:76–78. D’Souza VN, Nguyen TM, Morris GE, Karges W, Pillers DA, Ray PN. A novel dystrophin isoform is required for normal retinal electrophysiology. Hum Mol Genet 1995;4:837–842. Lidov HG, Selig S, Kunkel LM. Dp140: a novel 140 kDa CNS transcript from the dystrophin locus. Hum Mol Genet 1995;4:329– 335. Byers TJ, Lidov HG, Kunkel LM. An alternative dystrophin transcript specific to peripheral nerve. Nat Genet 1993;4:77–81. Hugnot JP, Gilgenkrantz H, Vincent N, et al. Distal transcript of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues. Proc Natl Acad Sci USA 1992;89:7506–7510. Gramolini AO, Dennis CL, Tinsley JM, et al. Local transcriptional control of utrophin expression at the neuromuscular synapse. J Biol Chem 1997;272:8117–8120. Matsumura K, Yamada H, Shimizu T, Campbell KP. Differential expression of dystrophin, utrophin and dystrophin-associated proteins in peripheral nerve. FEBS Lett 1993;334:281–285. Schofield J, Houzelstein D, Davies K, Buckingham M, Edwards YH. Expression of the dystrophin-related protein (utrophin) gene during mouse embryogenesis. Dev Dyn 1993;198:254–264.

S88

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89

[57] Roberts RG, Freeman TC, Kendall E, et al. Characterization of DRP2, a novel human dystrophin homologue. Nat Genet 1996;13:223–226. [58] Blake DJ, Schofield JN, Zuellig RA, et al. G-utrophin, the autosomal homologue of dystrophin Dp116, is expressed in sensory ganglia and brain. Proc Natl Acad Sci USA 1995;92:3697–3701. [59] Nguyen TM, Helliwell TR, Simmons C, et al. Full-length and short forms of utrophin, the dystrophin-related protein. FEBS Lett 1995;358:262–266. [60] Getman DK, Mutero A, Inoue K, Taylor P. Transcription factor repression and activation of the human acetylcholinesterase gene. J Biol Chem 1995;270:23511–23519. [61] Li Y, Camp S, Rachinsky TL, Bongiorno C, Taylor P. Promoter elements and transcriptional control of the mouse acetylcholinesterase gene. J Biol Chem 1993;268:3563–3572. [62] Perkins KJ, Burton EA, Davies KE. The role of basal and myogenic factors in the transcriptional activation of utrophin promoter A: implications for therapeutic up-regulation in Duchenne muscular dystrophy. Nucleic Acids Res 2001;29:4843–4850. [63] Santoro IM, Yi TM, Walsh K. Identification of single-strandedDNA-binding proteins that interact with muscle gene elements. Mol Cell Biol 1991;11:1944–1953. [64] Koike S, Schaeffer L, Changeux J-P. Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene. Proc Natl Acad Sci USA 1995;92:10624–10628. [65] Lev AA, Feener CC, Kunkel LM, Brown Jr. RH. Expression of the Duchenne’s muscular dystrophy gene in cultured muscle cells. J Biol Chem 1987;262:15817–15820. [66] Nudel U, Robzyk K, Yaffe D. Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain. Nature 1988;331:635–638. [67] Worton R. Muscular dystrophies: diseases of the dystrophin–glycoprotein complex. Science 1995;270:755–756. [68] Buonanno A, Rosenthal N. Molecular control of muscle diversity and plasticity. Dev Genet 1996;19:95–107. [69] Burden SJ. The formation of neuromuscular synapses. Genes Dev 1998;12:133–148. [70] Crowder CM, Merlie JP. Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines. Mol Cell Biol 1988;8:5257–5267. [71] Duclert A, Changeux JP. Acetylcholine receptor gene expression at the developing neuromuscular junction. Physiol Rev 1995;75:339– 368. [72] Hall ZW, Sanes JR. Synaptic structure and development: the neuromuscular junction. Cell 1993;72:99–121. [73] Prody CA, Merlie JP. The 5 0 -flanking region of the mouse muscle nicotinic acetylcholine receptor beta subunit gene promotes expression in cultured muscle cells and is activated by MRF4, myogenin and myoD. Nucleic Acids Res 1992;20:2367–2372. [74] Sanes JR, Hall ZW. Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J Cell Biol 1979;83:357–370. [75] Fromm L, Burden SJ. Synapse-specific and neuregulin-induced transcription require an ets site that binds GABPalpha/GABPbeta. Genes Dev 1998;12:3074–3083. [76] Rimer M, Cohen I, Lomo T, Burden SJ, McMahan UJ. Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle. Mol Cell Neurosci 1998;12:1–15. [77] Gramolini AO, Angus LM, Schaeffer L, et al. Induction of utrophin gene expression by heregulin in skeletal muscle cells: role of the Nbox motif and GA binding protein. Proc Natl Acad Sci USA 1999;96:3223–3227. [78] Gramolini AO, Burton EA, Tinsley JM, et al. Muscle and neural isoforms of agrin increase utrophin expression in cultured myotubes via a transcriptional regulatory mechanism. J Biol Chem 1998;273:736–743.

[79] Khurana TS, Rosmarin AG, Shang J, Krag TO, Das S, Gammeltoft S. Activation of utrophin promoter by heregulin via the ets-related transcription factor complex GA-binding protein alpha/beta. Mol Biol Cell 1999;10:2075–2086. [80] Fromm L, Burden SJ. Neuregulin-1-stimulated phosphorylation of GABP in skeletal muscle cells. Biochemistry 2001;40:5306–5312. [81] Sapru M, Florance S, Kirk C, Goldman D. Identification of a neureguin and protein-tyrosine phosphatase response element in the nicotinic acetylcholine receptor epsilo subunit gene: Regulatory role of an Ets transcription factor. Proc. Natl. Acad. Sci. USA 1998;95:1289–1294. [82] Schaeffer L, Duclert N, Huchet-Dymanus M, Changeux JP. Implication of a multisubunit Ets-related transcription factor in synaptic expression of the nicotinic acetylcholine receptor. EMBO J 1998;17:3078–3090. [83] Gegonne A, Bosselut R, Bailly RA, Ghysdael J. Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1. EMBO J 1993;12:1169–1178. [84] Rosmarin AG, Luo M, Caprio DG, Shang J, Simkevich CP. Sp1 cooperates with the ets transcription factor, GABP, to activate the CD18 (beta2 leukocyte integrin) promoter. J Biol Chem 1998;273:13097–13103. [85] Galvagni F, Capo S, Oliviero S. Sp1 and Sp3 physically interact and co-operate with GABP for the activation of the utrophin promoter. J Mol Biol 2001;306:985–996. [86] Sgouras DN, Athanasiou MA, Beal Jr. GJ, Fisher RJ, Blair DG, Mavrothalassitis GJ. ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation. EMBO J 1995;14:4781–4793. [87] Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987;51:987– 1000. [88] Edmondson DG, Olson EN. A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev 1989;3:628–640. [89] Wright WE, Sassoon DA, Lin VK. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 1989;56:607– 617. [90] Braun T, Gearing K, Wright WE, Arnold HH. Baculovirusexpressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer. Eur J Biochem 1991;198:187–193. [91] Rhodes SJ, Konieczny SF. Identification of MRF4: a new member of the muscle regulatory factor gene family. Genes Dev 1989;3:2050– 2061. [92] Jia HT, Tsay HJ, Schmidt J. Analysis of binding and activating functions of the chick muscle acetylcholine receptor gamma-subunit upstream sequence. Cell Mol Neurobiol 1992;12:241–258. [93] Piette J, Bessereau JL, Huchet M, Changeux JP. Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor alpha-subunit gene. Nature 1990;345:353–355. [94] Gramolini A, Jasmin B. Expression of the utrophin gene during myogenic differentiation. Nucleic Acids Res. 1999;27:3603–3609. [95] Biesiada E, Hamamori Y, Kedes L, Sartorelli V. Myogenic basic helix–loop–helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter. Mol Cell Biol 1999;19:2577–2584. [96] Donoviel DB, Shield MA, Buskin JN, Haugen HS, Clegg CH, Hauschka SD. Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice. Mol Cell Biol 1996;16:1649–1658. [97] Lassar AB, Buskin JN, Lockshon D, Davis RL, Apone S, Hauschka SD, Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell 1989;58:823–831.

K.J. Perkins, K.E. Davies / Neuromuscular Disorders 12 (2002) S78–S89 [98] Gong XQ, Li L. Dermo-1, a multifunctional basic helix–loop–helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation. J Biol Chem 2002;277: 12310–12317. [99] Hoogerwaard EM, de Voogt WG, Wilde AA, et al. Evolution of cardiac abnormalities in Becker muscular dystrophy over a 13year period. J Neurol 1997;244:657–663. [100] Marshall P, Chartrand N, Worton RG. The mouse dystrophin enhancer is regulated by MyoD, E-box-binding factors, and by the serum response factor. J Biol Chem 2001;276:20719–20726. [101] Bastianutto C, Bestard JA, Lahnakoski K, et al. Dystrophin muscle enhancer 1 is implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy. Hum Mol Genet 2001;10:2627–2635. [102] Galvagni F, Oliviero S. Utrophin transcription is activated by an intronic enhancer. J Biol Chem 2000;275:3168–3172. [103] Petrof B, Ebihara S, Guibinga G, et al. Presented at the American Society of Gene Therapy. Washington, DC: 1999. [104] Gilbert R, Nalbantoglu J, Petrof BJ, et al. Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles. Hum Gene Ther 1999;10:1299–1310. [105] Wakefield PM, Tinsley JM, Wood MJ, Gilbert R, Karpati G, Davies KE. Prevention of the dystrophic phenotype in dystrophin/utrophindeficient muscle following adenovirus-mediated transfer of a utrophin minigene. Gene Ther 2000;7:201–204. [106] Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992;89:5547–5551. [107] Ahmad A, Brinson M, Hodges BL, Chamberlain JS, Amalfitano A. Mdx mice inducibly expressing dystrophin provide insights into the potential of gene therapy for Duchenne muscular dystrophy. Hum Mol Genet 2000;9:2507–2515. [108] Hoffbrand A, Pettit J. Essential haematology. 2nd ed 1984. [109] Faller DV, Perrine SP. Butyrate in the treatment of sickle cell disease and beta-thalassemia. Curr Opin Hematol 1995;2:109–117. [110] Hudgins WR, Fibach E, Safaya S, Rieder RF, Miller AC, Samid D. Transcriptional upregulation of gamma-globin by phenylbutyrate

[111]

[112]

[113]

[114] [115]

[116]

[117]

[118]

[119]

[120]

[121]

S89

and analogous aromatic fatty acids. Biochem Pharmacol 1996;52: 1227–1233. Perrine SP, Dover GH, Daftari P, et al. Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo. Br J Haematol 1994;88:555–561. Perrine SP, Miller BA, Faller DV, et al. Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia. Blood 1989;74:454–459. Ikuta T, Kan YW, Swerdlow PS, Faller DV, Perrine SP. Alterations in protein-DNA interactions in the gamma-globin gene promoter in response to butyrate therapy. Blood 1998;92:2924–2933. Cappellini MD, Graziadei G, Ciceri L, et al. Butyrate trials. Ann N Y Acad Sci 1998;850:110–119. Perrine SP, Ginder GD, Faller DV, et al. A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders [see comments]. N Engl J Med 1993;328:81–86. Tinsley J, Deconinck N, Fisher R, et al. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med 1998;4:1441–1444. Corbi N, Libri V, Fanciulli M, Tinsley JM, Davies KE, Passananti C. The artificial zinc finger coding gene ‘Jazz’ binds the utrophin promoter and activates transcription. Gene Ther 2000;7:1076–1083. Blottner D, Luck G. Just in time and place: NOS/NO system assembly in neuromuscular junction formation. Microsc Res Tech 2001;55:171–180. Chaubourt E, Fossier P, Baux G, Leprince C, Israel M, De La Porte S. Nitric oxide and l-arginine cause an accumulation of utrophin at the sarcolemma: a possible compensation for dystrophin loss in Duchenne muscular dystrophy. Neurobiol Dis 1999;6:499–507. Chaubourt E, Voisin V, Fossier P, Baux G, Israel M, De La Porte S. Muscular nitric oxide synthase (muNOS) and utrophin. J Physiol Paris 2002;96:43–52. Carvajal JJ, Cox D, Summerbell D, Rigby PW. A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development. Development 2001;128:1857–1868.