Characterisation of expression of mDMAHP, a homeodomain-encoding gene at the murine DM locus
Characterisation of expression of mDMAHP , a homeodomain-encoding gene at the murine DM locusStephanie K. Heath+, Simon Carne, Christine Hoyle, Keith J. Johnson1 and Dominic J. Wells*
Gene Targeting Unit, Departments of Pharmacology and Clinical Neuroscience, Charing Cross & Westminster Medical School, London W6 8RF, UK and 1Division of Molecular Genetics,IBLS, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK
Received December 11, 1996;Revised and Accepted February 5, 1997
We examined the expression of the murine homologue of myotonic dystrophy associated homeodomain protein (mDMAHP) using two different strategies. The first approach, RT-PCR, detected spliced transcripts in a wide range of embryonic and adult tissues, in a pattern overlapping substantially with the expression of mDMPK. A second approach, the generation of transgenic mice expressing the lacZ reporter gene from a 4.3 kb promoter fragment, also demonstrated expression in a range of tissues with potential links to the phenotype in myotonic dystrophy. We conclude that murine DMAHP has a similar pattern of expression to human DMAHP and will serve as a useful model for functional studies of this gene, although species differences, such as the reduced CpG island (1.8 kb compared with 3.5 kb) must be carefully considered.
Myotonic dystrophy (DM) is an autosomal dominant disorder with an incidence of 1 in 8000 in most populations (1 ). It is characterised by myotonia, muscle weakness and wasting, but there are also disturbances in a number of other systems including the nervous system. The mutational mechanism is the progressive amplification of a CTG repeat at 19q13.3 (2 -7 ), resulting in a gradation of symptom severity and age of onset depending on the size of the repeat inherited. In unaffected individuals the repeat number ranges from 5-37 (8 ), whereas at least 50 copies are present in DM patients. The most severe form of the disorder is a congenital form, in which babies are born with severe hypotonia, respiratory distress and mental retardation. They may inherit repeat sizes in excess of 1500, which are almost always transmitted through an affected mother (9 ).
The CTG repeat lies in the 3' untranslated region of a protein kinase gene (DMPK), which is expressed primarily in skeletal and cardiac muscle, with lower levels in a collection of tissues including brain, lung and pancreas (3 ,10 -14 ). Conflicting data have been published on the effect of the repeat expansion on levels of expression of DMPK. Both an increase (13 ) and a decrease in transcript levels have been reported (11 ,12 ,15 ,16 ), although the majority of data currently available point to a fall in polyA+ RNA (17 ,18 ) and protein levels (11 ,16 ,19 ). How this would result in the dominant phenotype observed, however, is still unresolved.
The repeat also lies in the middle of a 3.5 kb CpG island, and this led recently to the discovery of a putative homeodomain-encoding gene (DMAHP) immediately 3' to DMPK (20 ). RT-PCR data have confirmed the presence of spliced transcripts of DMAHP in adult human tissues (20 ), but as yet there are little data on expression of DMAHP in mouse tissues. A partial mDMAHP cDNA clone (Six5) was recently isolated from a 1 month old mouse retina cDNA library, and was shown by in situ hybridisation to be weakly expressed in several layers of the mouse retina (21 ). This clone also confirmed the predicted intron-exon boundaries.
We have further compared murine and human DMAHP DNA sequence and note a marked difference in the size of the murine CpG island compared to that in the human. We have also examined the expression of murine DMAHP by RT-PCR, demonstrating the presence of spliced mDMAHP mRNA transcripts in a panel of both adult and fetal tissues. Additionally, a 4.3 kb mDMAHP promoter fragment was able to direct expression of the lacZ reporter gene in a wide range of embryonic tissues in several lines of transgenic mice. Our data suggest that mDMAHP is expressed in a variety of tissues during development, and also in the adult.
The murine CpG island at the 3' end of DMPK was found to be much less extensive than that in the human, being only 1.8 kb in size compared to 3.5 kb. The difference in size is mostly at the 5' end, with the murine island extending 5' only as far as exon 15 of DMPK, compared to that in the human which extends to intron 12 (Fig. 1 ). Interestingly, the murine CpG island does not include the site homologous to the CTG repeat in the human.
RT-PCR analysis of murine DMAHP expression was carried out using the primer pair MSO1F and KJDME2R on a range of adult and embryonic tissues. RT-PCR using a homologous set of primers on human DMAHP transcript produced a 1.1 kb fragment (20 ). The same sized fragment was obtained initially from RT-PCR on RNA derived from the heads of 12.5 day post coitum (d.p.c.) mouse embryos, and was sequenced to confirm that it was derived from a spliced mDMAHP gene transcript. As in humans this fragment consists of three exons, termed A, B and C, with A being the most 5' exon and C being the most 3' exon. The forward primer (MSO1F) binds in the homeodomain coding region located within exon A, and the reverse primer (KJDME2R) binds within exon C.
Expression of this spliced mDMAHP transcript was detected in a wide range of adult tissues including skeletal muscle, heart, testes, brain, smooth muscle, thymus, kidney and liver. Similarly, expression was detected in heads, limbs, liver, kidney and heart samples from 12.5 d.p.c. embryos (Fig. 2 a, b). RT-PCR studies of human tissues also showed in most cases a 337 bp fragment arising from alternative splicing, in which exon B was removed (20 ). An equivalent sized band was observed in adult mouse liver (Fig. 2 a) but cloning and sequencing of this band showed that it was an artefact. A band of ~500 bp was also observed in some of the murine samples examined but not on a consistent basis. This band was also cloned and sequenced and was again an artefact arising from the PCR reaction and was unrelated to mDMAHP.
As an internal control for the quality of each RNA preparation, RT-PCR amplification of mDMPK transcript was also carried out, using the primer pair CWROO3 (forward) and CWROO4 (reverse), designed to amplify a 233 bp fragment from exon 2 to exon 4 of DMPK. RT-PCR of the same set of adult and embryonic mouse tissues as used for the mDMAHP analysis detected the expected 233 bp product in all tissues assayed (Fig. 2 c, d). The relatively weak signal detected in the adult spleen and lung samples suggests that the absence of a clear mDMAHP PCR product may be due to inhibition of the RT or PCR reactions.
The CpG island is considerably less extensive in the mouse compared to the human. This difference is consistent with the report suggesting that the mouse genome has had a more rapid loss of CpG islands over evolutionary time than the human. It has been estimated that ~20% of human CpG islands are absent in the homologous mouse genes (22 ). The differences in DMAHP-associated CpG islands between the two species is also noteworthy given the position of the CTG repeat region, raising the possibility that mice engineered to carry expanded repeats at the site homologous to the human CTG expansion may not show identical effects on gene expression from the DM locus.
The RT-PCR data shown in Figure 2 demonstrates that mDMAHP is transcribed in a wide variety of different mouse tissues, both during development (12.5 d.p.c. embryos), and in the adult. Similar RT-PCR studies with human tissues also revealed expression in all tissues tested including skeletal muscle, brain, heart, fibroblasts and lymphoid cells (20 ). The comparison of the mDMPK expression patterns provides a degree of internal control for the mDMAHP PCR reactions. In general the patterns conform to that expected, both mDMPK and mDMAHP appear to be similarly expressed in a wide range of tissues. Although there appeared to be reproducible differences in the relative intensity of signals between the two genes in specific tissues, the lack of quantification with this technique means that such comparisons are of limited significance. Sequencing of the 1.1 kb mDMAHP fragment shows that it conforms to the same exon/intron boundaries as DMAHP, and also the partial murine cDNA clone Six5 (21 ). The high homology between mouse and human DMAHP, and the similar expression patterns derived from RT-PCR studies strongly suggest that studies of expression in mice will be useful for characterising the possible role of this gene in humans.
The transgenic studies demonstrate that there is significant promoter activity in the 4.3 kb promoter fragment, with expression of the lacZ reporter being detected in three independent transgenic lines. Patterns of expression were partially overlapping between the three lines although there was considerable variation in the intensity and timing of expression. Differences between the lines may be due to the strong position effect that the lacZ reporter can exhibit, even in the presence of strongly tissue specific promoters such as the [beta]-globin locus control region (23 ). Although the pattern of expression of the transgene did overlap with the expression pattern from the RT-PCR studies the match was clearly incomplete. Particularly noticeable was the absence of transgene expression in the heart. There are several possible reasons for this discrepancy. Firstly RT-PCR is a very sensitive method and may reveal expression at levels that will not be detected by visual examination following [beta]-galactosidase histochemistry. Secondly, although the transgenic approach is superior to RT-PCR of tissues, in that expression in specific cell types can be distinguished, the precise patterns of expression can depend heavily on the promoter fragment used to produce the mice. In these studies elements directing expression in cardiac tissues could be missing from the construct. However, despite the differences, it is clear that there is a functional promoter 5' to the homeodomain of mDMAHP.
The tissues that did express the transgene are interesting when considered in the context of pathological changes associated with myotonic dystrophy, the human disease associated with this locus. Cataract formation is one of the few symptoms in late onset myotonic dystrophy and we observed expression of the transgene in the lens capsule. Facial weakness is another feature of the disease and we observed transgene expression in the trigeminal and facial cranial nerves. Expression of the transgene is also detected in the vagus, which supplies parasympathetic innervation to abdominal and thoracic structures, including the heart. Cardiac, pharynx, oesophagus, stomach, small bowel, colon and sphincter abnormalities are reported in myotonic dystrophy often accompanied by dysphagia and abdominal pain (1 ). However such observations may prove to be coincidental once the function of the gene product is more clearly determined.
Other members of the mammalian Six gene family have already been characterised. Six1 and Six2 expression is observed in head and body mesenchyme, limb muscles and tendons (24 ). Six3 is expressed in the anterior neural plate and the developing eye (25 ). Six4 (AREC3) has been identified as a transcription factor regulating the Na+, K+-ATPase [alpha]1 subunit gene (Atpla1) and is expressed mainly in developing skeletal muscle. Weak expression of Six4 mRNA has also been reported for the heart, lung, kidney and brain (26 ). Six2, Six3a, Six3b and Six5 (mDMAHP) mRNAs are all expressed in specific layers of the retina (21 ). The expression patterns of mDMAHP revealed in the current study overlap with other members of the Six family but are not identical.
The identification of a gene downstream of DMPK, in close proximity to the CTG repeat and the CpG island, suggests that the repeat expansion may affect the expression of more than one gene. A number of possible mechanisms could be postulated for a role of DMAHP in the aetiology of DM. Enlarged CTG repeats have been shown to be strong nucleosome positioning elements (27 ), repeats of 75 and above being the strongest known natural nucleosome positioning elements so far discovered. Binding of nucleosomes could alter the local chromatin structure, and hence inhibit the passage of the transcription complex and repress the transcription of DMAHP. The presence of a DNaseI hypersensitive site has been demonstrated 3' of the triplet repeat in human fibroblasts and skeletal muscle (28 ). In three DM individuals there was overall DNaseI resistance in the region. The hypersensitive site maps to two PvuII recognition sites in a putative promoter region of DMAHP. Alternatively, CTG repeat expansion could result in methylation changes in the CpG island, and consequent downregulation of transcription. However, a previously reported study failed to detect any methylation differences between patients and normals (29 ). It is likely, therefore, that any changes in chromatin structure are independent of methylation status.
CTG repeats have also been shown to act as negative regulatory elements in the promoter regions of several genes. For example, in the mouse dioI gene, a 21 bp insert in intron two containing a (CTG)5 repeat results in decreased expression of the gene (30 ). In the murine growth inhibitory factor/metallothionein III gene promoter, a (CTG)25 repeat has repressive activity and was also shown to function as a silencer in vitro when coupled to promoters from other genes (31 ). Possible haploinsufficiency of DMAHP could account for some of the DM phenotype. Six5 has been shown to bind to the same DNA element as several other Six family gene products, and so could compete for this binding (21 ). Relatively minor changes in levels of DMAHP protein would then be critical in determining the co-ordination of key developmental processes or their maintenance in adult tissues. There are now a number of other autosomal dominant inherited conditions that arise through a haploinsufficiency of transcription factors or cofactors, for example PAX3 is implicated in Waardenburg's syndrome type 1 (32 ,33 ), PAX6 in aniridia (34 ), HOXD13 in synpolydactyly (35 ), TBX5 in Holt-Oram syndrome (36 ,37 ) and TWIST in Saethre-Chotzen syndrome (38 ,39 ). It is also possible that the CpG island itself could act as a locus control region, influencing the expression of other genes in addition to DMAHP. Clearly, further studies of DMAHP expression and investigations into the function of this homeodomain containing gene are required to examine any possible role in the pathogenesis of myotonic dystrophy. Ultimately, confirmation would be required through studies of expression in physiologically relevant tissues in patients.
The two approaches taken in this study to elucidate the expression of mDMAHP reflect the current lack of good antibodies for this gene. As these become available it should be possible to further define the expression patterns of both mDMAHP and DMAHP. The role of DMAHP is not currently known but, following the results obtained in the present study that show that mDMAHP has a functional promoter and that spliced transcripts are produced during embryogenesis and in adult life, knockout mouse experiments are underway to examine the functional importance of mDMAHP.
CpG island analysis of mDMPK and mDMAHP (accession numbers Z38015 and X84814 respectively) was carried out according to the criteria of Gardiner-Garden and Frommer (40 ). Moving average values for %(C+G) and for the CpG observed/expected (O/E) statistic were calculated using a 100 bp window, moving along the sequence at 50 bp intervals. CpG islands were defined as regions larger than 200 bp with moving averages %(C+G) >50% and CpG O/E >0.6.
Samples were collected from 12.5 d.p.c. F1 (C57Bl/10*CBA/J) embryos and a 4 month old C57Bl/10 male, snap frozen in liquid nitrogen, then thawed and homogenized in 2 ml of Trizol (Gibco) for every 100 mg of tissue. Manufacturers' instructions were followed for the recovery of total RNA. The RNA pellet was resuspended in 100 [mu]l DEPC-treated water and concentration determined on a spectrophotometer. All samples were DNaseI treated.
All reverse transcription (RT) reactions were performed using the Stratagene RT-PCR kit. Reactions were carried out according to the manufacturers' instructions using random primers. cDNA aliquots corresponding to 500 ng RNA equivalents were used as a template for each PCR reaction.
Amplification of the mDMAHP transcript was carried out using primers MSO1F (5' TGT GGA CAA ATA CCG GCT GC 3'), binding within the homeodomain region of exon A, and KJDME2R (5' TGA GGA TGA TCT TGC CCT GC 3'), binding within exon C. DNA was amplified in 50 [mu]l reaction volumes containing 400 mM each dNTP, 25 pmol each primer and 1.25 U Taq polymerase (Gibco) all in the supplied 1* reaction buffer containing 2 mM MgCl2, and overlaid with mineral oil. The reactions were denatured for 5 min at 95oC, at which stage the Taq polymerase was added, followed by 35 cycles of 95oC/1 min, 60oC/1 min, 72oC/3 min.
Amplification of the mDMPK transcript was carried out using primers CWROO3 (5' GAA TTC AGG CTT AAG GAG GTC CGA CTG 3'), binding in exon 2, and CWROO4 (5' GAA TTC GCA AAG TGC AGC TGT GTG ATC 3'), binding in exon 4 of murine DMPK. DNA was again amplified in 50 [mu]l reaction volumes containing 400 mM each dNTP, 25 pmol each primer and 1.25 U Taq polymerase (Gibco) all in the supplied reaction buffer containing 1.5 mM MgCl2, and overlaid with mineral oil. The reactions were again denatured for 5 min, followed by 3 cycles of 94oC/1 min 50 s, 61oC/1 min, 72oC/2 min, and 30 cycles of 94oC/1 min, 63oC/1 min 50 s, 72oC/2 min, and one cycle of 94oC/2 min, 61oC/2 min, and 72oC/7 min.
All PCR products were analysed by electrophoresis on a 1% agarose gel in 1* TBE buffer.
Construct pJB5DMAHP1 used for the production of transgenic mice was assembled from plasmid pJB5 (41 ) by the following steps. A 7.6 kb SalI-HindIII from bacteriophage S15-9 (described in 20 ) was subcloned into pBluescript (SK+) (Stratagene) and named 7.6SH2. The 2.0 kb KpnI rat actin promoter fragment was deleted from pJB5, and the resulting clone called pJB5K1. A 4.3 kb NotI-NruI fragment (the NotI site is derived from the polylinker of SK+) was excised from clone 7.6SH2, blunt-ended with Klenow, and ligated into pJB5K1 cut with Asp718 and blunt-ended with Klenow. The promoter-lacZ junction was sequenced to confirm correct orientation of the promoter fragment. The 7.9 kb promoter-lacZ fragment was excised with XbaI for linear transgenic injection fragment preparation.
Transgenic mice were produced by standard pronuclear injection of fertilised C57Bl/10 * CBA/J F1 eggs using a 1-2 ng/ml solution of XbaI linearised DNA which had been purified by electroelution and resuspended in 10 mM Tris, 0.1 mM EDTA pH 7.4. Eggs were cultured overnight and two cell embryos were transferred into pseudopregnant females of the same background. Screening of offspring was carried out by slot blot analysis of genomic tail DNA as described below.
Tail genomic DNA was purified by standard procedures (42 ). For slot blot analysis 2 [mu]g of DNA from each sample was denatured at 90oC in 0.4 M NaOH before being blotted onto Hybond N+ (Amersham). Membranes were hybridised with an alkaline phosphatase-labelled lacZ probe (from plasmid pJB5), and hybridisation detected using the Fluorescein Gene Images kit (Amersham). Controls of known copy number of plasmid in genomic DNA were also included to estimate copy number of the mice.
Control and hemizygous F1 embryos were recovered from pregnant females between 8.5 and 14.5 d.p.c. Embryos were dissected out of the embryonic membranes, washed in phosphate buffered saline (PBS) and fixed in 2% paraformaldehyde, 0.25% glutaraldehyde in PBS overnight at 4oC. Embryos were then washed three times for 30 min in PBS containing 1 mM MgCl2. Embryos were stained overnight at 37oC in a solution of 1 mM X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mM MgCl2 in PBS. After staining, embryos were washed in PBS then photographed. Transgenic embryos were dehydrated, cleared in histoclear and wax embedded. Sections of 8 [mu]m were cut, dewaxed, counterstained with alcoholic eosin and mounted with DPX. The identity of specific structures were confirmed using an atlas of mouse development (43 ).
We thank Dr E. Asante for the gift of pJB5. This work was funded by the Wellcome Trust and the Muscular Dystrophy Group of Great Britain and Northern Ireland.
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42 Hogan, B., Constantini, F. and Lacey, E. (1986) Manipulating the mouse embryo. Cold Spring Harbour Press.
43 Kaufman, M.H. (1992) The Atlas of Mouse Development. Academic Press.
*To whom correspondence should be addressed. Tel: +44 181 846 7038; Fax: +44 181 846 7377; Email: d.wells@cxwms.ac.uk
+Present address: Molecular Analysis of Mammalian Mutation, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
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