PANTALLA “ESTADÍSTICA_AGUA”

A striking feature of dystrophin transcription is the encodement of multiple isoforms transcribed from different promoters (Fig 1.3). Three and maybe four promoters regulate the expression of three 427kDa “full-length” isoforms and four internal promoters drive the expression of four COOH- terminal short isoforms. The structural homology with utrophin was

emphasised by finding that the position of at least one, and perhaps three, of these promoters was conserved between the two genes. The structure, expression and function of the dystrophin isoforms is considered in sections

1.5, 1.6 and 1.7 in order to allow comparison with the utrophin forms bearing in mind the evolutionary relationship between these two proteins.

M u scle is o fo rm The muscle full-length dystrophin is transcribed from a promoter situated c. 310Kb from dystrophin exon 2 and is active in skeletal, cardiac and smooth muscle and in brain (Chelly et al., 1990).

B rain is o fo rm The brain specific isoform, also known as cortical or cerebral dystrophin or C-dystrophin (Nudel et al., 1989), is transcribed from a promoter 90Kb upstream from the muscle promoter and 400Kb from exon 2 (Boyce et al., 1991). This transcript is tightly restricted to cortical and hippo­ campal neuronal cells and contains a unique first exon encoding 344 bp of

Dp260 Dp140 Dpi 16 Dp71

NH

cysteine-rich COOH _Full-length

0=3 M=11 P=7 NH cysteine-rich COOH Dp260 cysteine-rich NH COOH _{Dpi 40} NH cysteine-rich COOH _Dp116 Dp71 cysteine-rich COOH NH

Fig 1.3 Upper. The positions of the transcription start sites of the 8 known dystrophin mRNAs are shown relative to their protein structure. Lower. The structure of each isoform. The NHg-terminal domains are coloured blue if they contain novel aa encoded by the first exon; the number of aa is indicated. In the case of Dp140, coloured grey, the first exon encodes only 5’ UTR. The four NHg- isoforms shown are L= Lymphoid, C= Cerebral, M= Muscle and P=Purkinje.

5’ UTR and 3 aa which are different from those encoded by the muscle transcript exon 1 (Chelly et al., 1990; Lidov et al., 1990; Bies et al., 1992).

Cerebellar isoform A full-length form with a novel first exon

encoding 270bp of 5’ UTR and a novel NH2 terminus comprising 7 aa is

expressed only in Purkinje cells of the cerebellum. The position of the first exon of the Purkinje specific transcript lies in the middle of the intron between

muscle-type exon 1 and exon 2 (Gorecki et al., 1992; Abdulrazzak et al., 2001).

Lymphoid isoform A further full-length isoform of dystrophin (L-

dystrophin) was identified in cultured lymphoblastoid cells from a DMD patient with a deletion extending from the 5’ end of the gene to exon 2 (Nishio et al., 1994). The position of the promoter for L-dystrophin was mapped more than 500Kb upstream of the brain promoter, thereby adding 500Kb to the size of the dystrophin gene (Nishio et al., 1994). This novel first exon encodes 46 bp of 5’ UTR which are spliced directly to exon 3 with the creation of a possible ATG initiation codon within exon 3 (Nishio et al., 1994). There have been no further studies of this isoform.

There is now good evidence for selective mechanisms that regulate the expression of full-length dystrophin from these alternative 5’ promoters. For example, in cardiac muscle, in contrast to skeletal muscle, only the muscle specific promoter is active. As a consequence, mutations (and deletions) at the 5’ end of the DMD gene in the region of the muscle promoter result in a

progressive heart disease without skeletal muscle myopathy (Muntoni et al., 1993, 1995; Milasin et al., 1996; Ferlini et al., 1999). This disorder, X-linked

dilated cardiomyopathy (XLDC), is allelic to DMD. In the skeletal muscle of affected males full-length dystrophin is transcribed, by a compensatory

mechanism, from the brain and purkinje specific promoters allowing the muscle to escape the dystrophic changes (Milasin et al., 1996, Muntoni et al., 1995).

In addition to the three full-length transcripts described above the

dystrophin gene locus also contains internal promoters that regulate expression of at least four short transcripts, Dp71, D pi 16, D p i40 and Dp260. The protein products of these transcripts do not have the NH2-terminal region but share the

same COOH-terminal and cysteine-rich domains and in some cases part of the long central rod like domain (Fig 1. 3). These isoforms were identified as

unusual bands on Western or Northern blots and their cDNAs were recovered either by deliberate screening of tissue specific cDNA libraries or by 5’ RACE.

Dp71 isoform A short isoform of dystrophin, Dp71, was initially

identified as a 6.5 Kb dystrophin transcript on Northern blots. Transcription of Dp71 is initiated approximately 8Kb upstream from the intron62/exon 63 boundary (Hugnot et al., 1992; Lederfein et al., 1992) and the transcript

encodes a 5' UTR of 80 nt and 7 novel aa spliced to exon 63 (Fig 1.3). The size of the transcript is 4.8 Kb (not 6.5Kb as initially estimated by Northern blotting) and the protein, with a molecular weight of 70.8kDa, contains the cysteine rich and carboxy-terminal domains. The isoform was designated Dp71 to reflect its molecular weight.

Dp71 mRNA has been detected by Northern blotting in several tissues including brain, liver, testis, lung and kidney and cell lines such as Schwannoma

and glioma C6 cells (Bar et al., 1990; Blake et al., 1992). Dp71 polypeptide has been detected by Western blotting in a similar range of tissues and cell lines and shows some developmental regulation; for example, Dp71 is expressed in foetal skeletal muscle but not in adult muscle (Blake et al., 1992; Hugnot et al., 1992; Schofield et al., 1994; Loh et al., 2000). The expression of Dp71 mRNA during mouse embryonic development was analysed more fully by mRNA in situ

hybridisation using a riboprobe specific for the unique first exon (Schofield et al., 1994). Dp71 mRNA was detected at 11.5 dpc in the floor-plate and in the whole length of the neural tube by 12.5 dpc. It is also expressed in condensing

mesenchyme in regions of the maxilla and mandible, in developing teeth and in liver. In the neonate brain, Dp71 mRNA is found in the ventral midbrain and caudate putamen. In an independent study, Dp71 mRNA was detected in

glomeruli, tubules and collecting ducts of the developing kidney as well as in the developing eye (Durbeej et al., 1997).

Antibodies raised against different regions of the dystrophin protein have been used to determine the subcellular localisation of Dp71. In these studies the use of dystrophin deficient mice, mdx, has been particularly useful. The findings can be summarised in brief as follows; in foetal skeletal muscle Dp71 is expressed at the sarcolemma of myotubes before the “switching-on” of full- length dystrophin but its expression declines towards birth (Tennyson et al., 1996; Lin S. et al., 2000); Dp71 is the most abundant dystrophin isoform in retina (Rodius et al., 1997; Arsanto et al., 1999) where it localises to the outer and inner plexiform and nuclear layers and to the cytoplasm of Müller cells (Rodius et al., 1997; Claudepierre et al., 2000). In the brain, Dp71 is located in the choroid plexii, ependymal lining of the ventricles, olfactory bulb.

hippocampal regions and cerebral cortex. In the cerebellum, Dp71 is found in the molecular, granular and Purkinje layers in glial cells (Lambert et al., 1993; Jung et al., 1993; Gorecki et al., 1995). This isoform is also found in glial cells of the cerebral cortex in contrast to full-length dystrophin which is expressed only in neurones (Blake et al., 1999).

D pi 16 isoform A second dystrophin short isoform. D pi 16, is

transcribed from a promoter within intron 55 (Byers et al., 1993). The D pi 16 novel first exon contains more than 200 nucleotides of 5’ UTR and coding sequence for 23 unique aa (Fig 1.3) (Schofield et al., 1994). D p i 16 protein (1 16kDa) consists of three spectrin repeats, the cysteine rich domain and the carboxy-terminal domain.

Northern blot analysis showed that expression is confined to glioma. Schwannoma and oligodendroglioma cells (Byers et al., 1993; Schofield et al.,

1994). D pi 16 is a rare transcript during embryogenesis as judged by mRNA in situ hybridisation experiments using a D pi 16 specific probe; mRNA was only weakly detected in the liver by 12.5 dpc and in brain after birth (Schofield et al., 1994).

In adults. D pi 16 protein is expressed in peripheral nerves and immuno- histochemical analysis showed that D pi 16 localises to a thin rim around the outside of the myelinated fibre (Byers et al., 1993). It also appears to be

expressed in the inner ear. Western blots of control and m dx mice cochlea and immuno-histochemistry have shown that D pi 16 is expressed in both types of hair cells in the cochlea alongside full-length dystrophin; in these cells D pi 16 predominates at synaptic regions (Dodson et al., 1995).

Dp140 isoform The 140kDa polypeptide, Dp140, is encoded by a 7.5Kb mRNA transcribed from a promoter within intron 44. A unique non-coding first exon (110bp) is spliced to exon 45 (Lidov et al., 1995). Dp140 contains the last 6 repeats of the rod domain, the cysteine-rich and the COOH-terminal domains of the dystrophin protein (Fig 1.3) (Lidov et al., 1995).

Expression of D p i40 protein is restricted to foetal kidney, retina and brain (Lidov et al., 1998; Rodius et al., 1997); in the latter. D p i40 protein accumulates in the olfactory bulb and leptomeningeal surfaces (Lidov et al., 1995). The expression of D p i40 mRNA and protein in the developing kidney has been analysed in some detail by mRNA in situ hybridisation, using a D p i40 specific probe, and by immuno-histochemistry, using several anti-dystrophin COOH-terminal antibodies, (Durbeej et al., 1997). By 15 dpc, Dp140 mRNA and protein accumulate in the comma-shaped tubules at the basal side of the

epithelial cells next to the basal membrane, no expression is detected in collecting ducts or glomeruli. D p i40 is absent from the adult kidney which indicates that in this tissue this isoform is most important during morphogenesis (Durbeej et al., 1997).

Dp260 isoform The identification of the Dp260 isoform was

prompted by clinical observations concerning DMD patients. Some patients have an ocular phenotype characterised by an abnormal electroretinogram (ERG) with a reduced b-wave amplitude (Fillers et al., 1993; Fitzgerald et al., 1994). This is suggestive of abnormal signal transmission across the outer plexiform layer (GPL) of the retina and it was of interest to find an associated loss of dystrophin immunoreactivity at the post-synaptic region of the GPL in

eye sections from these patients (Piliers et al., 1993, Miike et al., 1989; Tamura et al., 1993). The observation that mdx mice have a normal ERG b-wave while the mouse has a dramatically reduced b-wave, implicated an isoform transcribed from a promoter situated between exon 23 (position of the mutation in mdx mice) and 65 (position of the mutation in mdx ^^^).

This prediction was confirmed by the detection of a novel 260kDa band on Western blots of mouse retina extract which could be detected using an antibody raised against an exon 32 fusion protein whereas an antibody against an exon 28 protein did not detect this isoform (D’Souza et al., 1995). The corresponding mRNA was recovered by 5’ RACE and sequence analysis revealed that the transcriptional start site was likely to lie within intron 29. The unique first exon contains 21 Ib p of 5’ UTR and 13 aa and is spliced to exon 30 (D’Souza et al., 1995). The Dp260 isoform is the longest of the short

dystrophins and consists of 15 spectrin repeats, 2 hinge regions and the

cysteine and COOH-terminal domains (Fig 1.3). Western blot analysis of retinal extracts prepared at different stages of mouse development showed that Dp260 protein levels increase perinatally (Rodius et al., 1997). Reverse transcription FOR (RT-PCR) indicates that the Dp260 transcript is expressed strongly in retina and weakly in brain and heart, with no expression in a variety of other somatic tissues (D’Souza et al., 1995).

The expression profile of the different dystrophin isoforms was reviewed by Tokarz et al. (1998) using RT-PCR; full-length dystrophin mRNA and the short transcripts were amplified from a wide selection of mouse adult tissues suggesting that their expression might be wider than previously estimated by Northern and/or Western blotting. This finding was perhaps not surprising since

RT-PCR is a very sensitive technique and trace amounts of mRNA may be amplified.

In summary, there is firm evidence that the short dystrophin isoforms are transcribed and translated in numerous tissues; while Dp71 is widely

distributed, expression of Dp116, Dp140 and Dp260 seems more restricted. In all four cases, the patterns of mRNA and protein expression show some

correlation.

The expression pattern of the dystrophin isoforms is summarised in Table 1.1.