Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (10)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Murakami, Y.
Right arrow Articles by Kawakami, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murakami, Y.
Right arrow Articles by Kawakami, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 2103-2112  

Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements
Introduction
Results
   Expression of mDMAHP/Six5 mRNA in various tissues, various stages of the embryo and P19 cells
   Identification of transcription initiation sites of mDMAHP/Six5
   Analysis of the regulatory region of the mDMAHP/Six5 promoter
   Identification of elements and binding factors for the positive/negative regulatory region
   Factor binding and regulatory function of the elements
Discussion
Materials And Methods
   Construction of plasmids
   Cell culture
   Transfection and luciferase and [beta]-galactosidase assays
   RNA isolation and northern blot analysis
   RNase protection and primer extension assays
   Gel retardation assays
   Methylation interference analysis
Acknowledgements
References

Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements

Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements

Yoshiaki Murakami1,2, Hiromi Ohto1, Uichi Ikeda2, Kazuyuki Shimada2, Takashi Momoi3 and Kiyoshi Kawakami1,*

1Department of Biology and 2Department of Cardiology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan and 3Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Tokyo, Japan

Received June 26, 1998; Revised and Accepted September 14, 1998

We analyzed the expression of mouse DMAHP/Six5 (the myotonic dystrophy-associated homeodomain protein gene) during embryogenesis and in various tissues by northern blotting. Expression was observed as early as embryonic day 7 (E7) and continued to E17. Abundant expression was observed in neonatal heart and skeletal muscle with potential links to the phenotype of myotonic dystrophy. The transcription initiation sites of the gene were analyzed in mouse E11 and E15 embryos and in adult skeletal and heart muscle. Three major transcription initiation sites were identified, the proximal site was specific to the early E11 embryo, while the other two were common among the heart and skeletal muscle and E11 and E15 embryos. All transcription initiation sites were downstream of the corresponding CTG repeat locus of the mouse gene (-1195), excluding a possible inclusion of the CUG repeat sequence in mRNA leading to abnormal splicing or to translation of aberrant protein. For analysis of the regulatory elements in the promoter region, we used P19 embryonal carcinoma cells which abundantly express mouse DMAHP/Six5. Multiple positive and negative elements were identified in the promoter region. All positive elements were Sp1/Sp3 binding sites and one of the negative elements was a novel factor binding site. The transcription initiation sites and regulatory elements are conserved between human and mouse DMAHP.

INTRODUCTION

mDMAHP/Six5 is a mouse homolog of the myotonic dystrophy-associated homeodomain protein (DMAHP) gene (1). It is also known as one of the members of the Six family genes, which are mouse homologs of the Drosophila sine oculis gene (2). Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by muscle weakness and wasting and disturbances in the nervous system (3). The causative CTG repeat is located in the 3[prime]-non-coding region of DMPK, which is upstream of DMAHP, and expansion of the CTG repeat induces the neuromuscular disorder (4). Progressive amplification of the repeat results in a gradation of the severity of symptoms and age of onset. CTG repeat expansion in this locus alters the adjacent chromatin structure and may cause repression of the surrounding genes (5). Contradictory results have been published regarding the expression of surrounding genes. The nuclear retention of DMPK mRNA but not DMAHP mRNA was observed in fibroblast cells of patients with DM (6,7), while expression of the DM-linked DMAHP allele but not DMPK was reduced in myoblasts, muscle and myocardium from the patients (8,9). DMPK gene knockout mice show mild myopathy or late onset progressive myopathy, but never show myotonia, which is the hallmark of DM (10,11). Therefore, a reduction in DMAHP expression is thought to be involved in DM, although it may not be the only underlying mechanism of the disease.

The expression of DMAHP and mDMAHP is observed in a wide variety of adult tissues, including skeletal and heart muscles, where the main symptoms of DM are observed. Such expression is demonstrated by RT-PCR analysis of RNA isolated from human and mouse tissues (1,12). The presence of DMAHP/Six5 protein in nuclei of various adult tissues has been identified by gel retardation assay using nuclear extracts from rats (13). The expression of mDMAHP was observed in a range of tissues with potential links to the phenotype in DM in the embryo of transgenic mice which contained a 4.3 kb mDMAHP promoter fragment fused to the lacZ reporter gene.

Six family genes are probably involved in differentiation and morphogenesis. This is based on the observation that Six1, Six2, Six3 and Six4 show a temporally and spatially restricted expression pattern in mouse, chicken and zebrafish embryos (13-17). Furthermore, overexpression of the zebrafish six3 gene is associated with enlargement of the rostral forebrain in zebrafish (18) and so mutants show defects in complex eye formation in Drosophila (19,20). Because the Six2, Six4 and Six5 gene products share a similar DNA binding specificity, the complex network among Six family genes must be involved in the development and morphogenesis process (2,21).

Analysis of DMAHP/Six5 expression and the regulatory mechanism of gene expression is indispensable for our understanding of the biological role of the gene in development and also for an understanding of the development of DM. In the present study, we examined the transcription initiation sites of mDMAHP/Six5 using embryonic and adult tissue RNAs and identified the regulatory elements in the promoter region by transient transfection assays using P19 embryonal carcinoma cells as a model system.

RESULTS

Expression of mDMAHP/Six5 mRNA in various tissues, various stages of the embryo and P19 cells

The expression of DMAHP has been observed in a wide variety of tissues, including skeletal and cardiac muscle, brain and fibroblast cell lines (1). Expression of its mouse homolog mDMAHP has also been detected in a wide variety of embryonic and adult tissues (12). Such analyses were based on RT-PCR but gene expression has not been analyzed quantitatively. For this purpose, we analyzed the expression of mDMAHP/Six5 mRNA by northern blotting. In the adult mouse, mDMAHP/Six5 mRNA was expressed strongly in the heart, moderately in the lung, kidney and liver and weakly in skeletal muscle, spleen and testis (Fig. 1A). The expression of mDMAHP/Six5 mRNA in neonatal heart and skeletal muscle was much stronger than other tissues (Fig. 1B). In embryos, mDMAHP/Six5 mRNA was detected as early as embryonic day 7 (E7). The level of expression increased and it was still present at E17 (Fig. 1C). In every tissue, multiple species of mRNAs were observed, varying in size from ~4.3 to 2.9 kb.

   A

   B
   C

   D

Figure 1. Northern blotting of mDMAHP/Six5 mRNA. (A) Mouse MTN blot. Mouse tissues are listed above each lane. Positions of RNA size markers are shown to the left. (B) Aliquots of 2 µg poly(A)+ RNA from adult heart (lane 1) and skeletal muscle (lane 2) and neonatal heart (lane 3) and skeletal muscle (lane 4) were analyzed. Positions of 28S and 18S rRNA are indicated. (C) Mouse embryo MTN blot. Embryonic days are shown above the lanes. (D) Aliquots of 5 (lanes 1-7) or 4.5 µg (lanes 8-19) poly(A)+ RNA from P19 cells were analyzed. M, monolayer culture (lane 1); A2, A4, days 2 and 4 of aggregation culture with (lanes 3 and 5) or without (lanes 2 and 4) treatment with retinoic acid; D2, day 2 of differentiation culture after aggregate formation (lanes 6 and 7); M1, M3, days 1 and 3 of monolayer culture with (lanes 9 and 11) or without (lanes 8 and 10) treatment with retinoic acid; A, aggregation culture (lanes 12 and 13); D1, D3, D5, days 1, 3 and 5 of differentiation culture (lanes 14-19); RA+, addition of retinoic acid during the monolayer or aggregation culture period. No retinoic acid was added in the differentiation culture.

Since mDMAHP/Six5 mRNA was strongly expressed in embryos and mDMAHP/Six5 is thought to play a role in the pathophysiology of DM during development, we used the P19 embryonal carcinoma (teratocarcinoma) cell line and examined expression of mDMAHP/Six5 mRNA. P19 cells can differentiate into neuronal cells following treatment with retinoic acid and aggregation culture. To examine the possible involvement of mDMAHP/Six5 in this process, we analyzed gene expression by northern blotting. Expression of mDMAHP/Six5 mRNA in P19 cells was weak under growth conditions (Fig. 1D, lane 1). Treatment with 1 µM retinoic acid and aggregation culture resulted in overexpression of mDMAHP/Six5 mRNA (lanes 3 and 5) compared with control cultures in the absence of retinoic acid (lanes 2 and 4). On the other hand, expression decreased during a 2 day culture in differentiation medium, to a level comparable with retinoic acid-free control cultures (lanes 6 and 7).

We also investigated whether the induction of mDMAHP/Six5 mRNA was due only to retinoic acid or both retinoic acid and aggregation culture. For this purpose, we used a modified protocol in which the cells were first treated with retinoic acidand were then transferred to aggregation medium. The addition of 1 µM retinoic acid to monolayer cultures inducedmDMAHP/Six5 mRNA expression (lanes 9 and 11). Furthermore, overexpression persisted in aggregation culture (lane 13). However, expression gradually decreased during 3-5 days culture in differentiation medium (lanes 15, 17 and 19). These results indicate that induction of mDMAHP/Six5 occurred through retinoic acid and probably in the early differentiation process into neuronal cells.

The control northern analysis with a [beta]-actin probe showed approximately the same degree of signal in each lane (data not shown).

Identification of transcription initiation sites of mDMAHP/Six5

In order to identify the transcription initiation site in mouse tissues, we first performed RNase protection assays on poly(A)+ RNA isolated from neonatal heart and skeletal muscle tissues in which mDMAHP/Six5 mRNA expression was most abundant. Using an antisense RNA probe containing an HaeIII fragment (Fig. 2A) which covers the translation initiation site, only 152 nt signals corresponding to the whole region of mRNA in the probe were detected (Fig. 2A, lanes 1 and 2, arrow b). Another RNA probe (AluI fragment) which covers the translation initiation site also yielded the protected signal corresponding to the whole mRNA region (data not shown). These results indicate that the transcription initiation site resides upstream of the probe. To locate the site, we used a further upstream RNA probe (EcoRI-AluI fragment). The 263 nt signals corresponding to the whole region of mRNA in the probe and 228 nt signals, which indicate the transcription initiation site within the probe region, were observed both in the heart and skeletal muscle (Fig. 2B, lanes 1 and 2, arrows b and a, respectively). The major transcription initiation site indicated by the 228 nt signal was 371 bp upstream of the A of the initiating methionine codon. Another four signals below the 228 nt signal were observed and indicated transcription initiation sites within a 25 bp region downstream of this major site. We named these five sites the intermediate initiation sites (see below). The 263 nt signal corresponding to the full-length protected one on heart and skeletal muscle RNAs indicates a further upstream transcription initiation site. To locate the site, we performed a primer extension assay, which detected the distal transcription initiation site (Fig. 2C, lanes 3 and 4). This was 520 bp upstream of the A of the initiating methionine codon. The distal transcription initiation site was also detected in adult heart and skeletal muscle (Fig. 2C, lanes 1 and 2).

   A
   B
   C

Figure 2. Identification of transcription initiation sites of mDMAHP/Six5. (A) RNase protection assay of RNAs from various tissues with the probe including the HaeIII region of mDMAHP/Six5. Aliquots of 2 µg poly(A)+ RNA from neonatal heart (lane 1) and skeletal muscle (lane 2), P19 cells with (lane 4) or without (lane 3) retinoic acid treatment, E11 (lane 5) and E15 (lane 6) embryos were analyzed. Non-digested probe (lane 7) and control hybridization with yeast RNA (lane 8) are also shown. The position of size markers is shown to the right. a, 127 nt signal; b, 152 nt signal corresponding to the whole mRNA region of the probe; c, 237 nt signal corresponding to the full size of the probe. (B) RNase protection assay with the probe including the EcoRI-AluI region. RNAs used are as in (A). a, 228 nt signal; b, 263 nt signal corresponding to whole mRNA region of the probe; c, 322 nt signal corresponding to the full size of the probe. Bracket shows the additional four signals within the 25 bp region. (C) Primer extension assay. Aliquots of 2 µg poly(A)+ RNA were analyzed. The samples are from adult heart (lane 1) and skeletal muscle (lane 2) and neonatal heart (lane 3) and skeletal muscle (lane 4), P19 cells with (lane 6) or without (lane 5) treatment with retinoic acid and E11 (lane 7) and E15 (lane 8) embryos. M, size marker (lane 9).

To determine whether the transcription initiation site of the mDMAHP/Six5 gene in the embryo was the same as in the adult tissue, we performed a series of RNase protection and primer extension assays using poly(A)+ RNA from E11 and E15 whole embryos and the same set of probes and primers as above. RNase protection assays with the proximal primer (HaeIII) identified 152 nt signals corresponding to the whole mRNA region on both E11 and E15 RNAs. On the other hand, a shortened protected signal of 127 nt was found only on the RNA from E11 (Fig. 2A, lanes 5 and 6). This new signal indicated a proximal transcription initiation site, which was located 93 bp upstream of the A of the initiating methionine codon and was set as +1. Using another proximal probe (AluI), the same transcription initiation site was again detected on E11 RNA (data not shown). Analysis with the upstream probe (EcoRI-AluI) showed the same signal of 228 nt and an additional four signals within a 25 bp downstream region as on heart and skeletal muscle RNAs (Fig. 2B, lanes 5 and 6). The most upstream intermediate transcription initiation site was estimated as -278. The primer extension assay showed that the distal transcription initiation site was similar to those on the RNAs of heart and skeletal muscle, which was estimated as position -427.

For the proximal (+1) and intermediate multiple initiation sites (-278 to -253), we carried out primer extension analyses (data not shown). However, only prematurely terminated transcripts were observed. These results were probably due to the high GC content in these regions. The possibility that the proximal initiation site (+1) represents a splice acceptor site from a distant upstream start site or alternative first exon has not been ruled out.

To identify the exact transcription initiation sites utilized in embryonal carcinoma P19 cells, we also performed RNase protection and primer extension assays on poly(A)+ RNA from P19 cells. The above-mentioned three transcription initiation sites were all demonstrated on P19 cell RNA irrespective of treatment with retinoic acid (Fig. 2A, lanes 3 and 4; B, lanes 3 and 4; and C, lanes 5 and 6). All transcription initiation sites were similar to those of the E11 embryos. P19 cells were used in the present study for analysis of mDMAHP/Six5 promoter elements since the transcription initiation sites in early mouse embryos are conserved in this cell line.

Analysis of the regulatory region of the mDMAHP/Six5 promoter

To identify the regulatory elements of the mDMAHP/Six5 promoter in the mouse, we performed reporter gene assays using mDMAHP/Six5-luciferase chimeric constructs in P19 cells cultured without retinoic acid treatment. We measured luciferase activity of a nested set of mDMAHP/Six5 5[prime] flanking deletion constructs. Shortening the upstream sequence to nt -683 did not significantly change luciferase activity (data not shown). For the -683 to -50 sequential 5[prime] deletion constructs there were several regions that significantly inhibited or augmented luciferase activity. Shortening the upstream sequence from -434 to -344 resulted in a 74% reduction in luciferase activity. Further deletion to -317 resulted in a 2.4-fold increase and another deletion to -270 resulted in 60% inhibition of activity. Shortening the sequence from -224 to -178 resulted in a 3.3-fold increase in activity, while deletion from -137 to -97 resulted in an 81% decrease in activity (Fig. 3). These results indicate that the positive elements reside between -434 and -344, -317 and -270, and -137 and -97. The negative elements reside between -344 and -317, and -224 and -178.


Figure 3. Reporter gene assays of 5[prime] sequential deletion constructs of the mDMAHP/Six5 promoter. Each of the plasmids (3 µg) was co-transfected with pEF-BOS[beta]GAL (0.3 µg) into P19 cells by the CaPO4 method. The cells were cultured for 24-48 h without retinoic acid treatment. Luciferase activity was normalized with respect to the [beta]-galactosidase activity in the same cell lysate. The relative activity of pSix5-683LF was expressed as 100. Values are means ± SEM.

When these reporter gene assays were performed with retinoic acid treatment, luciferase activities in each construct were slightly increased to roughly the same extent. Elements responsive to retinoic acid have not been identified.

Identification of elements and binding factors for the positive/negative regulatory region

To examine whether any transcription factor binds to the regulatory region identified above, we performed gel retardation assays using nuclear extracts from P19 cells without retinoic acid treatment. We prepared five probes containing regulatory regions suggested by the transient transfection assays. Each of three probes that contained putative positive elements (-434 to -344, -317 to -270, and -137 to -50) gave three retarded complexes (Fig. 4, lanes 1, 4 and 7, C1, C2 and C3). The pattern of formation of retarded complexes was very similar among these three probes and they were suspected to be Sp1/Sp3 complexes based on the nucleotide sequence in the probes. A DNA fragment containing an Sp1 consensus sequence competed with formation of these complexes (data not shown). The formation of C2 was inhibited by addition of anti-Sp1 antibody and a supershifted complex was observed (Fig. 4, lanes 2, 5 and 8), while formation of C1 and C3 was inhibited by addition of anti-Sp3 antibody (Fig. 4, lanes 3, 6 and 9). Thus, all three positive regulatory regions contained Sp1/Sp3 binding elements.


Figure 4. Gel retardation assays of the positive regulatory regions of the mDMAHP/Six5 promoter. Five to ten femtomoles of 32P-labeled fragments of mDMAHP/Six5 (-434 to -344 in lanes 1-3, -317 to -270 in lanes 4-6, and -137 to -50 in lanes 7-9) were incubated with a nuclear extract from P19 cells without retinoic acid treatment (1.8 µg protein). C1, C2 and C3 indicate the positions of the retarded complexes. Aliquots of 300 ng of anti-Sp1 (lanes 2, 5 and 8) or anti-Sp3 (lanes 3, 6 and 9) antibodies were added.

Gel retardation assays with the probe for the -224 to -178 region gave four retardation complexes (Fig. 5, lane 1). C1-C3 proved to be Sp1/Sp3 complexes by gel retardation assay using specific anti-Sp1 and anti-Sp3 antibodies (data not shown). To determine whether C4 was a sequence-specific binding complex, we added a competitor DNA fragment from -202 to -174 to the gel retardation assay. Formation of C4 was inhibited but that of C1-C3 was not, indicating that the binding region of C4 resides within -202 to -178 (Fig. 5, lane 2). In the next step, we examined the binding sequence of C4 using the methylation interference assay. Seven guanine residues in the sequence GGGAAACTGAGG between -189 and -178 were protected (Fig. 5B and C). Searches of the profile database of the transcription factor binding site (22) showed no known factor recognizing this sequence. Thus, this negative regulatory element is a novel transcription factor binding site.

   A
   B

   C

Figure 5. Gel retardation and methylation interference assays of the proximal negative regulatory region. (A) Gel retardation assay. Four femtomoles of the 32P-labeled fragment of mDMAHP/Six5 (-224 to -178) was incubated with a nuclear extract from P19 cells (1.8 µg protein) (lane 1). Aliquots of 400 fmol double-stranded DNA oligomer (-202 to -174) was added as competitor (lane 2). C1, C2, C3 and C4 indicate the positions of the sequence-specific retarded complexes observed. Other complexes were non-specific. (B) Methylation interference assay. The gel retardation complex C4 (C) and unretarded probe (F) were isolated and analyzed (lanes 2-3 and 5-6). G residues of the C4 complex that are interfered with are indicated by arrowheads. Maxam-Gilbert guanine ladders (G) are shown (lanes 1 and 4). (C) Summary of the methylation interference assay. G residues that are interfered with are indicated by arrowheads.

Gel retardation assay with a probe for the -344 to -317 region gave no retardation complexes. Although this region functions as a negative element, factor-DNA binding may not be visualized, at least under our experimental conditions.

Findings on gel retardation complex formation with nuclear extracts from P19 cells with retinoic acid treatment were the same as those without retinoic acid treatment. Thus, the factors binding to these elements identified by gel retardation assay were not regulated by retinoic acid.

Factor binding and regulatory function of the elements

To confirm that the factor binding sites identified above function as regulatory elements of the mDMAHP/Six5 promoter, we prepared three mutation constructs pSix5-434mLF, pSix5-317mLF and pSix5-224mLF, in which Sp1/Sp3 sites at -434 to -344, -317 to -270 and negative element -189 to -178 were mutated. The promoter activity was measured by transient transfection assay. pSix5-434mLF and pSix5-317mLF showed reduced luciferase activities compared with those of the corresponding wild-type promoter constructs (76 and 32% of that of pSix5-434LF and pSix5-317LF, respectively). On the other hand, pSix5-224mLF showed increased luciferase activity (2.9-fold) compared with pSix5-224LF (Fig. 6). These results indicate that the Sp1/Sp3 binding elements from -366 to -359 and -309 to -302 act as positive elements and the negative factor binding site from -189 to -178 certainly acted as a negative element. Luciferase activities of pSix5-317mLF and pSix5-224mLF were comparable with those of pSix5-270LF and pSix5-178LF, respectively. Thus, reduced or enhanced promoter activities between -317 and -270 and between -224 and -178 are explained by the elements identified. However, luciferase activity of pSix5-434mLF was higher than that of pSix5-344LF, indicating that mutation did not completely abolish the enhancing activity between -434 and -344. We tested the binding activity of the corresponding factor to the mutated sequence by gel retardation competition assay. Mutations in pSix5-317mLF and pSix5-224mLF resulted in complete loss of binding activity of Sp1/Sp3 and the negative regulatory factor, respectively (data not shown). A mutation in pSix5-434mLF, however, retained weak binding activity of Sp1/Sp3 (data not shown). A careful examination of the sequence showed two overlapping Sp1/Sp3 binding sites (Fig. 7, -366 to -357 and -359 to -350) and that mutation abolished only one of the Sp1/Sp3 binding sites. These overlapping Sp1/Sp3 sites, which showed positive regulatory effects, have also been observed in other genes, such as the Na,K-ATPase [alpha]2 subunit gene (23).


Figure 6. Reporter gene assays of point mutations in the positive/negative regulatory elements. Each of the plasmids (1 µg) was co-transfected with pEF-BOS[beta]GAL (0.1 µg) into P19 cells using LipofectAMINE PLUS reagent. Luciferase activity based on the relative activity of pSix5-434mLF was set at 100. Values are means ± SEM. Sp1/Sp3 binding elements are shown as open boxes and the negative factor binding elements are shown by dotted boxes. Mutated elements are shown.

   A
   B

Figure 7. (A) Summary of the mDMAHP/Six5 promoter elements and transcription initiation sites; comparison of the sequence with human DMAHP. The top line shows the mouse (M) sequence, whereas the bottom line shows the human (H) sequence. Asterisks indicate identity of bases at those positions. Arrows represent the transcription initiation sites. Human transcription initiation sites were identified by Klesert et al. (9). The most proximal transcription initiation sites in both sequences are numbered +1. Identified cis elements are shown by solid boxes. A tentative distal negative element is shown, which is suggested by the transfection study and sequence homology between human and mouse. The translation initiation codon (coding region) and the mouse sequence corresponding to human (CTG)n are also shown. (B) Positive and negative regulatory elements in 5[prime] sequential deletion constructs of mDMAHP/Six5. Sp1/Sp3 binding elements are shown by open boxes and the negative factor binding elements are shown by dotted boxes.

DISCUSSION

We have demonstrated an extensive sequence homology in the promoter regions between human and mouse DMAHP, from -835 to the translation initiation site +93 (Fig. 7). The transcription initiation sites of human DMAHP (9) have been identified at a similar location as the intermediate transcription initiation sites in mDMAHP, where several transcription initiation sites are clustered within ~25 bp. Considering the sequence gap around -245 in the mouse gene, the multiple transcription initiation sites of both DMAHP and mDMAHP are approximately equivalent distances from the proximal transcription initiation site (+1) in the mouse. Future analysis of human embryonic tissue may show the proximal initiation site in DMAHP as observed in mDMAHP. All three positive regulatory Sp1/Sp3 binding sites and the novel factor binding negative elements are conserved between human and mouse DMAHP. The HS region of human DMAHP (from -508 to -304) is thought to be an enhancer element, which showed an ~150-fold increase when fused to the heterologous HSV thymidine kinase promoter (9). We also tested the corresponding region in mDMAHP (from -811 to -520) for enhancer activity by fusion to the HIV-1 core promoter. Our results showed a 10- to 14-fold increase in promoter activity (data not shown). These conserved features in transcription initiation sites and regulatory elements strongly suggest that the regulatory mechanisms of both human and mouse DMAHP are conserved. This notion is also supported by the wide distribution of DMAHP mRNA in both human and mouse tissues (1,12).

Another major finding of the present study was the identification of expression of mDMAHP/Six5 as early as E7, which persisted during embryonic development. We also identified abundant expression of mDMAHP/Six5 in the heart and skeletal muscle of neonatal and adult mice. The major symptoms of DM are myotonia and heart failure in some cases, which are consistent with the above expression pattern of mDMAHP/Six5. We identified three major transcription initiation sites: the proximal site was specific to the early E11 embryo, while the other two were initiation sites common among heart and skeletal muscle and E11 and E15 embryos. Transcripts from different transcription initiation sites might encode alternatively spliced forms of mRNA. This should be clarified by a careful analysis of mRNA species during the early stages of embryogenesis. All of the transcription initiation sites are downstream of the corresponding CTG repeat position of the mouse gene (-1195). This rules out the possibility that the CUG repeat sequence is included in DMAHP mRNA and that it produces abnormally spliced mRNA or aberrant protein, such as polyleucine. Therefore, the effects of the CTG repeat on DMAHP must be solely at the transcriptional level. To test whether the expanded CTG repeat from DM patients affects expression of mDMAHP/Six5, we inserted CTG repeats of from 50 to 180 repeats in the corresponding region of the mouse gene and measured luciferase activity by transient transfection into P19 cells. Insertion of the CTG repeat resulted in marginal effects on luciferase activity (data not shown), suggesting an important effect of chromatin structure on transcription.

P19 cells are known to differentiate into neuronal cells following treatment with retinoic acid and aggregation culture. mDMAHP/Six5 was induced by a similar treatment (Fig. 1D). This suggests a possible involvement of mDMAHP/Six5 in neuronal differentiation of P19 cells. Because the Six5 gene product is a sequence-specific binding protein (2,13) and is expected to be a transcription factor, induction of mDMAHP/Six5 can modulate expression of its target genes involved in neuronal differentiation. In this context, a reduction in DMAHP mRNA in early embryogenesis of DM patients might lead to immature neuronal or muscle cells, resulting in the development of DM. Indeed, maturational delay of muscle cells has been observed in neonatal DM patients (24,25). As for the induction mechanism of the gene, a retinoic acid-responsive element (RARE) (26) sequence was not observed from the 5[prime]-flanking region (-3444) to the 3[prime] end of mDMAHP/Six5 (1). Therefore, the response element may reside outside this region or may be a sequence distinct from RARE.

Based on the 5[prime] deletion analysis, the region between -680 and +92 was sufficient for expression of the mDMAHP/Six5 gene in P19 cells. In this promoter region there is no TATA box but several Sp1/Sp3 consensus sequences are present, at least three of which were shown to be positive regulatory elements of the gene. Two negative regulatory elements were also identified in our experiments and at least one is a novel factor binding site (Figs 3, 6 and 7B). These analyses allow an understanding of the fundamental architecture of the mDMAHP/Six5 promoter which will form the basis for future analysis of development- and tissue-specific regulation of the promoter and the repression mechanism of transcription by the CTG repeat.

MATERIALS AND METHODS

Construction of plasmids

The mouse genomic clone containing the last four exons of DMPK (27) and part of the first exon of the mDMAHP/Six5 gene was isolated from a library (Stratagene). For the 5[prime]-promoter deletion analysis, a nested set of mDMAHP/Six5 promoter fragments was isolated by digestion with various restriction enzymes at the 5[prime] ends and digestion with NlaIII, which recognizes the translation initiation site at the 3[prime] end. These fragments were subcloned into luciferase reporter plasmid pSV0A/L[Delta]5[prime] (28). The restriction sites used in the present experiments were SacII (-1738), TaqI (-811), BglI (-683), PvuII (-508), FspI (-434), Sau3AI (-344), EcoRI (-317), ApaI (-270), Kpn2I (-224), EcoO109I (-178), HhaI (-137), SmaI (-97), AluI (-50) and HpaII (-6). The longest construct contained nucleotides from position -3444. The constructs were named pSix5-3444LF, pSix5-1738LF, pSix5-811LF, pSix5-683LF, pSix5-508LF, pSix5-434LF, pSix5-344LF, pSix5-317LF, pSix5-270LF, pSix5-224LF, pSix5-178LF, pSix5-137LF, pSix5-97LF and pSix5-50LF.

Point mutation constructs pSix5-434mLF and pSix5-224mLF were prepared using PCR-mediated mutagenesis. Synthetic oligonucleotides used were as follows. For pSix5-434mLF, 5[prime]-TAGCGCGATTCCAAGAACCTCCTCCCAGCTAG-3[prime] and 5[prime]-GCTGGGAGGAGGTTCTTGGAATCGCGCTAG-3[prime] (mutated nucleotides underlined). For pSix5-224mLF, 5[prime]-CTGATAGGGAAAATTATTCCCTGAGTCAGAGG-3[prime] and 5[prime]-TCTGACTCAGGGAATAATTTTCCCTATCAGAG-3[prime]. pSix5-317mLF was constructed using annealed oligonucleotides 5[prime]-AGCTTAATTCCGAGGTTCTTAGCACGGCGCGGAGATGGGAAGGGAGGGGGCC-3[prime] and 5[prime]-CCCTCCCTTCCCATCTCCGCGCCGTGCTAAGAACCTCGGAATTA-3[prime]. They were subcloned into the HindIII and ApaI site of pSix5-434LF.

[beta]-Galactosidase expression vector pEF-BOS[beta]GAL(29) was used as an internal control for luciferase gene expression.

Cell culture

P19 cells were maintained in [alpha] minimum essential medium ([alpha]-MEM; Gibco BRL) supplemented with 10% fetal calf serum as previously described (30). Cells were passaged at 2 day intervals at a ratio of 1:20. For induction experiments, P19 cells were treated in two ways. The first was according to the original procedure of McBurney et al. (31). P19 cells were treated with 1 µM retinoic acid and incubated on bacterial grade plastic dishes to facilitate aggregation. After 2-4 days, cells were washed and resuspended in [alpha]-MEM without retinoic acid and plated on tissue culture dishes and further incubated for 1-3 days. The second treatment was the modified method that distinguished the effects of retinoic acid stimulation and aggregation culture. P19 cells were treated with 1 µM retinoic acid and incubated on tissue culture dishes (monolayer culture). After 3 days, the cells were washed and resuspended in [alpha]-MEM without retinoic acid and incubated on bacterial grade dishes for 1 h (aggregation culture). Aggregation occurred during this period. The aggregates were plated onto tissue culture dishes and incubated for 3-5 days (differentiation culture).

Transfection and luciferase and [beta]-galactosidase assays

P19 cells were transiently transfected using the standard CaPO4 method (32) or LipofectAMINE PLUS reagent (Gibco BRL) and were analyzed 24-48 h after transfection. Luciferase and [beta]-galactosidase activities were assayed as previously described (33).

RNA isolation and northern blot analysis

The mouse and mouse embryo Multiple Tissue Northern (MTN) blots were purchased from Clontech. Total RNA was isolated from adult and 1-day-old ICR mice, whole E11 and E15 embryos and P19 cells using Isogen (Nippongene). Poly(A)+ RNA was isolated using an Oligo(dT)-Cellulose/Microfuge pack (Becton Dickinson) according to the instructions provided by the manufacturer. Northern blotting was performed as described (34), using 2 µg poly(A)+ RNA. The probe for hybridization was a 1485 bp NcoI-BglII fragment excised from mouse Six5 cDNA (2). The experimental protocol was approved by the Ethics Review Committees for Animal Experimentation of the participating institutions.

RNase protection and primer extension assays

RNase protection assay probes were prepared as follows. The 236 bp AluI fragment (-50 to +186) and 152 bp HaeIII fragment (-26 to +126) of the mDMAHP/Six5 genomic clone, which include a translation initiation site, and the 5[prime] flanking 267 bp EcoRI-AluI fragment (-317 to -51) were subcloned into pKS (Bluescript KS M13+). These plasmids were truncated with suitable enzymes and transcribed with T3 RNA polymerase in the presence of [32P]UTP. The resulting RNA was purified by urea PAGE followed by elution and was used for hybridization (35). The RNase protection assay was performed with the RPA II system (Ambion) according to the protocol provided by the manufacturer. Aliquots of 2 µg mouse tissue or P19 cell poly(A)+ RNA were hybridized overnight with excess amounts of 32P-labeled antisense probe. Following RNase digestion, protected products were resolved on 6% denaturing polyacrylamide gels and visualized by exposure to X-ray film.

Primer extension assays were performed using single-stranded DNA primers as described previously (36). The primer was designed to detect the distal transcription initiation site; the sequence was 5[prime]-GAGTGTCGGTCCAAAAAGCGG-3[prime]. An aliquot of 2 µg poly(A)+ RNA was used for each reaction.

Gel retardation assays

Nuclear extracts were prepared from P19 cells as described by Schreiber et al. (37). Gel retardation assays were performed as described previously (38). The FspI(-434)-Sau3AI(-344), Eco47I(-344)-EcoRI(-317), EcoRI(-317)-ApaI(-270), Kpn2I(-224)-EcoO109I(-178) or HhaI(-97)-AluI(-50) fragment was labeled with [[alpha]-32P]dCTP and used as the probe. Anti-Sp1 and anti-Sp3 polyclonal antibodies were purchased from Santa Cruz Biotechnology. As a competitor for the probe Kpn2I(-224)-EcoO109I(-178) fragment, we used an annealed oligonucleotide covering -202 to -174. The sequence of the upper strand was 5[prime]-TTGCTCTGATAGGGAAACTGAGGCC-3[prime].

Methylation interference analysis

Methylation interference analysis was performed as described previously (38). The probe for the reaction was prepared as follows. Oligonucleotide 5[prime]-TTGCTCTGATAGGGAAACTGAGGCC-3[prime] and its complementary sequence were annealed and subcloned into SmaI-digested pKS (Bluescript KS M13+). This plasmid was digested with XhoI and SacI. The XhoI site was 3[prime]-labeled with [[alpha]-32P]dCTP by Klenow fragment or 5[prime]-labeled with [[gamma]-32P]ATP by T4 polynucleotide kinase. These probes were partially methylated and used for the reaction.

ACKNOWLEDGEMENTS

This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan and by a JMS Graduate Student Research Award.

REFERENCES

1. Boucher, C.A., King, S.K., Carey, N., Krahe, R., Winchester, C.L., Rahman, S., Creavin, T., Meghji, P., Bailey, M.E., Chartier, F.L., Brown, S.D., Siciliano, M.J. and Johnson, K.J. (1995) A novel homeodomain-encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG)n repeat. Hum. Mol. Genet., 4, 1919-1925. MEDLINE Abstract

2. Kawakami, K., Ohto, H., Takizawa, T. and Saito, T. (1996) Identification and expression of six family genes in mouse retina. FEBS Lett., 393, 259-263. MEDLINE Abstract

3. Harris, S., Moncrieff, C. and Johnson, K. (1996) Myotonic dystrophy: will the real gene please step forward! Hum. Mol. Genet., 5, 1417-1423. MEDLINE Abstract

4. Fu, Y.H., Pizzuti, A., Fenwick, R.G. Jr, King, J., Rajnarayan, S., Dunne, P.W., Dubel, J., Nasser, G.A., Ashizawa, T., de Jong, P., Wieringa, B., Korneluk, R., Perryman, M.B., Epstein, H.F. and Caskey, C.T. (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science, 255, 1256-1258. MEDLINE Abstract

5. Otten, A.D. and Tapscott, S.J. (1995) Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. Proc. Natl Acad. Sci. USA, 92, 5465-5469. MEDLINE Abstract

6. Davis, B.M., McCurrach, M.E., Taneja, K.L., Singer, R.H. and Housman, D.E. (1997) Expansion of a CUG trinucleotide repeat in the 3[prime] untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc. Natl Acad. Sci. USA, 94, 7388-7393. MEDLINE Abstract

7. Hamshere, M.G., Newman, E.E., Alwazzan, M., Athwal, B.S. and Brook, J.D. (1997) Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes. Proc. Natl Acad. Sci. USA, 94, 7394-7399. MEDLINE Abstract

8. Thornton, C.A., Wymer, J.P., Simmons, Z., McClain, C. and Moxley, R.T. (1997) Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nature Genet., 16, 407-409. MEDLINE Abstract

9. Klesert, T.R., Otten, A.D., Bird, T.D. and Tapscott, S.J. (1997) Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nature Genet., 16, 402-406. MEDLINE Abstract

10. Reddy, S., Smith, D.B., Rich, M.M., Leferovich, J.M., Reilly, P., Davis, B.M., Tran, K., Rayburn, H., Bronson, R., Cros, D., Balice Gordon, R.J. and Housman, D. (1996) Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nature Genet., 13, 325-335. MEDLINE Abstract

11. Jansen, G., Groenen, P.J., Bachner, D., Jap, P.H., Coerwinkel, M., Oerlemans, F., van den Broek, W., Gohlsch, B., Pette, D., Plomp, J.J., Molenaar, P.C., Nederhoff, M.G., van Echteld, C.J., Dekker, M., Berns, A., Hameister, H. and Wieringa, B. (1996) Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nature Genet., 13, 316-324. MEDLINE Abstract

12. Heath, S.K., Carne, S., Hoyle, C., Johnson, K.J. and Wells, D.J. (1997) Characterisation of expression of mDMAHP, a homeodomain-encoding gene at the murine DM locus. Hum. Mol. Genet., 6, 651-657. MEDLINE Abstract

13. Ohto, H., Takizawa, T., Saito, T., Kobayashi, M., Ikeda, K. and Kawakami, K. (1998) Tissue and developmental distribution of Six family gene products. Int. J. Dev. Biol., 42, 141-148.

14. Oliver, G., Wehr, R., Jenkins, N.A., Copeland, N.G., Cheyette, B.N., Hartenstein, V., Zipursky, S.L. and Gruss, P. (1995) Homeobox genes and connective tissue patterning. Development, 121, 693-705. MEDLINE Abstract

15. Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A. and Gruss, P. (1995) Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development, 121, 4045-4055. MEDLINE Abstract

16. Bovolenta, P., Mallamaci, A., Puelles, L. and Boncinelli, E. (1998) Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors. Mech. Dev., 70, 201-203. MEDLINE Abstract

17. Seo, H.-C., Drivenes, O., Ellingsen, S. and Fjose, A. (1998) Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia. Mech. Dev., 73, 45-57. MEDLINE Abstract

18. Kobayashi, M., Toyama, R., Takeda, H., Dawid, I.B. and Kawakami, K. (1998) Overexpression of the forebrain-specific homeobox gene six3 induces rostral forebrain enlargement in zebrafish. Development, 125, 2973-2982. MEDLINE Abstract

19. Serikaku, M.A. and O'Tousa, J.E. (1994) sine oculis is a homeobox gene required for Drosophila visual system development. Genetics, 138, 1137-1150. MEDLINE Abstract

20. Cheyette, B.N., Green, P.J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S.L. (1994) The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron, 12, 977-996. MEDLINE Abstract

21. Kawakami, K., Ohto, H., Ikeda, K. and Roeder, R.G. (1996) Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development. Nucleic Acids Res., 24, 303-310. MEDLINE Abstract

22. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A.E., Kel, O.V., Ignatieva, E.V., Ananko, E.A., Podkolodnaya, O.A., Kolpakov, F.A., Podkolodny, N.L. and Kolchanov, N.A. (1998) Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res., 26, 362-367. MEDLINE Abstract

23. Ikeda, K., Nagano, K. and Kawakami, K. (1993) Anomalous interaction of Sp1 and specific binding of an E-box-binding protein with the regulatory elements of the Na, K-ATPase [alpha]2 subunit gene promoter. Eur. J. Biochem., 218, 195-204. MEDLINE Abstract

24. Sarnat, H.B. (1994) New insights into the pathogenesis of congenital myopathies. J. Child Neurol., 9, 193-201. MEDLINE Abstract

25. Iannaccone, S.T., Bove, K.E., Vogler, C., Azzarelli, B. and Muller, J. (1986) Muscle maturation delay in infantile myotonic dystrophy. Arch. Pathol. Lab. Med., 110, 405-411. MEDLINE Abstract

26. Perlmann, T., Rangarajan, P.N., Umesono, K. and Evans, R.M. (1993) Determinants for selective RAR and TR recognition of direct repeat HREs. Genes Dev., 7, 1411-1422. MEDLINE Abstract

27. Mahadevan, M.S., Amemiya, C., Jansen, G., Sabourin, L., Baird, S., Neville, C.E., Wormskamp, N., Segers, B., Batzer, M., Lamerdin, J., de Jong, P., Wieringa, B. and Korneluk, R.G. (1993) Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene. Hum. Mol. Genet., 2, 299-304. MEDLINE Abstract

28. de Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol., 7, 725-737. MEDLINE Abstract

29. Suzuki-Yagawa, Y., Kawakami, K. and Nagano, K. (1992) Housekeeping Na,K-ATPase [alpha]1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind. Mol. Cell. Biol., 12, 4046-4055. MEDLINE Abstract

30. Rudnicki, M.A. and McBurney, M.W. (1987) In Robinson, E.J. (ed.), Teratocarcinomas and Embryonic Stem Cells-A Practical Approach. IRL Press, Oxford, UK, pp. 19-49.

31. McBurney, M.W., Reuhl, K.R., Ally, A.I., Nasipuri, S., Bell, J.C. and Craig, J. (1988) Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture. J. Neurosci., 8, 1063-1073. MEDLINE Abstract

32. Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol., 7, 2745-2752. MEDLINE Abstract

33. Murakami, Y., Ikeda, U., Shimada, K. and Kawakami, K. (1997) Promoter of the Na,K-ATPase [alpha]3 subunit gene is composed of cis elements to which NF-Y and Sp1/Sp3 bind in rat cardiocytes. Biochim. Biophys. Acta, 1352, 311-324. MEDLINE Abstract

34. Watanabe, Y., Kawakami, K., Hirayama, Y. and Nagano, K. (1993) Transcription factors positively and negatively regulating the Na,K-ATPase [alpha]1 subunit gene. J. Biochem. Tokyo, 114, 849-855.

35. Kawakami, K., Suzuki Yagawa, Y., Watanabe, Y. and Nagano, K. (1992) Identification and characterization of the cis-elements regulating the rat AMOG (adhesion molecule on glia)/Na,K-ATPase [beta]2 subunit gene. J. Biochem. Tokyo, 111, 515-522.

36. Lillie, J.W., Green, M. and Green, M.R. (1986) An adenovirus E1a protein region required for transformation and transcriptional repression. Cell, 46, 1043-1051. MEDLINE Abstract

37. Schreiber, E., Matthias, P., Muller, M.M. and Schaffner, W. (1989) Rapid detection of octamer binding proteins with `mini-extracts', prepared from a small number of cells. Nucleic Acids Res., 17, 6419. MEDLINE Abstract

38. Kawakami, K., Scheidereit, C. and Roeder, R.G. (1988) Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-[kappa]B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro. Proc. Natl Acad. Sci. USA, 85, 4700-4704. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +81 285 58 7311; Fax: +81 285 44 5476; Email: kkawakam@jichi.ac.jp

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 13 Nov 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
Y. Ohinata, S. Sutou, M. Kondo, T. Takahashi, and Y. Mitsui
Male-Enhanced Antigen-1 Gene Flanked by Two Overlapping Genes Is Expressed in Late Spermatogenesis
Biol Reprod, December 1, 2002; 67(6): 1824 - 1831.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Sato, M. Nakamura, D. H. Cho, S. J. Tapscott, H. Ozaki, and K. Kawakami
Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1
Hum. Mol. Genet., May 1, 2002; 11(9): 1045 - 1058.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H. Ohto, S. Kamada, K. Tago, S.-I. Tominaga, H. Ozaki, S. Sato, and K. Kawakami
Cooperation of Six and Eya in Activation of Their Target Genes through Nuclear Translocation of Eya
Mol. Cell. Biol., October 1, 1999; 19(10): 6815 - 6824.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (10)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Murakami, Y.
Right arrow Articles by Kawakami, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murakami, Y.
Right arrow Articles by Kawakami, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?