| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
An L1 element intronic insertion in the black-eyed white (Mitfmi-bw) gene: the loss of a single Mitf isoform responsible for the pigmentary defect and inner ear deafness
Introduction
Results
Pigmentation phenotype of the Mitfmi-bw allele
Molecular lesion of the Mitfmi-bw allele
Effect of Mitfmi-bw on the expression of Mitf-A and Mitf-H
Functional analysis of the Mitf-H and Mitf-A variants
Effect of Mitfmi-bw on the expression of Mitf-M
Functional analysis of the effect of Mitf intron 3 on Mitf-M expression
Discussion
Effects of multiple promoters and L1bw insertion on Mitf isoform expression
Implications for human WS2 conditions
The role of Mitf in the development of melanocytes and RPE
L1 retrotransposition-induced mutations in humans and mice
Materials And Methods
Mice
Cells and transfection assays
Genomic DNA isolation and PCR analysis
RNA isolation and RT-PCR analysis
Plasmid clones
Acknowledgements
References
An L1 element intronic insertion in the black-eyed white (Mitfmi-bw) gene: the loss of a single Mitf isoform responsible for the pigmentary defect and inner ear deafness
Received January 22, 1999; Revised and Accepted May 5, 1999
Waardenburg syndrome type 2 (WS2) is an autosomal dominant disorder characterized by a combination of pigmentary and auditory abnormalities. Approximately 20% of WS2 cases are associated with mutations in the gene encoding microphthalmia-associated transcription factor (MITF). MITF plays a critical role in the development of both neural-crest-derived melanocytes and optic cup-derived retinal pigmented epithelium (RPE); the loss of a functional Mitf in mice results in complete absence of all pigment cells, which in turn induces microphthalmia and inner ear deafness. The black-eyed white Mitfmi-bw homozygous mouse normally has a pigmented RPE but lacks melanocytes essential for the pigmentation of the body and hearing. We show here that Mitfmi-bw is caused by an insertion into intron 3 of a 7.2 kb novel L1 element, L1bw, which belongs to an actively retrotransposing TF subfamily. The L1bw insertion reduces the amount of mRNAs for two Mitf isoforms, Mitf-A and Mitf-H, by affecting their overall expression levels and pre-mRNA splicing patterns, while it abolishes mRNA expression of another isoform, Mitf-M, which is specifically expressed in neural-crest-derived melanocytes. The consequence of the L1 insertion in the black-eyed white Mitfmi-bw mouse is that the developmental programme for RPE cells proceeds normally, most likely because of the presence of residual, full-length Mitf-A and Mitf-H proteins, whereas the lack of Mitf-M results in loss of the melanocyte population. The results suggest that melanocyte development depends critically on a single Mitf isoform, Mitf-M, and raise the possibility that specific mutations affecting MITF-M, the human equivalent of Mitf-M, may be responsible for a subset of WS2 conditions.
INTRODUCTION
The combination of abnormal pigmentation and hearing loss are found in most mammals. In humans, Waardenburg syndrome (WS), an autosomal dominant disorder, is one of the most frequent of such conditions (1 per 40 000 live births) identified by pigmentary abnormalities, white spotting, iris heterochromia and early greying, with sensorineural deafness (1). It has been shown that WS is clinically and genetically heterogeneous and classified into four types (WS1, WS2, WS3 and WS4). Mutations in the PAX3 gene account for the majority of WS1 and WS3 cases, while certain mutations in the EDN3, EDNRB and SOX10 genes can cause WS4 (2-5). For WS2, which is distinguished from WS1/3 by the absence of distopia canthorum, ~20% of cases are associated with mutations in the gene encoding the microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (6-8). MITF is essential for the development of two different types of pigment cells, neural-crest-derived melanocytes and optic cup-derived retinal pigmented epithelium (RPE). Mice homozygous for an apparent null allele (Mitfvga-9) at the orthologous microphthalmia (Mitf) locus show white coat and inner ear deafness (9,10), both of which are caused by the absence of melanocytes from the skin, hair follicles and the stria vascularis of the cochlea (11,12), and also severe microphthalmia, because of the defective RPE (13). In addition, MITF is able to specifically bind and activate the transcription of pigment cell-specific melanogenesis genes such as tyrosinase (TYR), TYRP1 and TYRP2 (14), that have within the promoters a CATGTG E-box motif flanked by a critical T residue (15). In one WS2 pedigree with a polymorphic TYR allele (TYRR402Q) encoding a tyrosinase enzyme with reduced catalytic activity, the occurrence of ocular albinism is associated with the presence of both the TYRR402Q allele and an unlinked MITF mutation (16), suggesting that some other hypopigmentation phenotypes found in WS2 patients may involve down-regulation of TYR gene expression.
Several MITF mutations have been shown to exhibit WS2 conditions in the heterozygous state. These include an in-frame deletion [del(R217)], amino acid substitutions (S250P, N278D and S298P), nonsense mutations (R259X and R214X), a 1 bp deletion resulting in a premature termination codon in the last coding exon and splice site mutations at the flanking intronic sequences predicted to produce truncated proteins (6,16-20). Although the mutation del(R217), one of the most severe alleles (17,19), is expected to encode a mutant MITF protein that acts in a dominant-negative fashion (10,21), other MITF mutations have been proposed to cause WS2 conditions due to haploinsufficiency of the normal MITF protein (18). In contrast, mutant alleles at the mouse Mitf locus, including those similar or equivalent to MITF mutations [truncating mutations and del(R217)], are inherited as recessive or semi-dominant traits (10,11,22-24). This difference can be explained, as mice are less sensitive than humans to gene dosage required for the normal function of Mitf protein in melanocyte development (18). It is possible that as yet unidentified human MITF mutations which correspond to some of the mouse Mitf alleles are responsible in the heterozygous states for a subset of WS2 patients. Understanding how these Mitf alleles cause pigmentary and hearing abnormalities may therefore provide insights into the pathophysiology of WS2 and the underlying biological processes.
In 1954, Kreinter discovered a recessive white spotting mutation (25) that was subsequently shown to be allelic with MitfMi-wh (26) and was termed black-eyed white (Mitfmi-bw). The Mitmi-bw allele is one of the oldest known among white spotting mutations in which the defect is demonstrated to lie within the precursor cells (27) and contributed to the conception of the `preprogrammed cell death' theory (28). When homozygous, Mitfmi-bw usually results in a complete black-eyed white phenotype with severe hearing loss (29) but without any ocular abnormalities. In contrast to most Mitf alleles, which reduce retinal pigmentation and induce microphthalmia and/or retinal degeneration, Mitfmi-bw appears to specifically disrupt neural-crest-derived melanocyte development. However, it is unknown whether the melanocyte-specific Mitfmi-bw phenotype reflects a difference in the dependency of precursor cells in the two pigment cell lineages on functional Mitf proteins.
Here we show that an intronic insertion of a L1 retrotransposable element in the Mitfmi-bw gene slightly reduces the amount of mRNAs for two Mitf isoforms, the RPE-enriched isoform (Mitf-A) (30) and the heart-type isoform (Mitf-H) (23), while it blocks the expression of mRNAs for the melanocyte-specific isoform, Mitf-M. The developmental programme for normally pigmented RPE is not impaired by these changes, whereas the lack of Mitf-M results in melanocyte loss in the black-eyed white Mitfmi-bw mouse. The results provide an explanation for the Mitfmi-bw phenotype and suggest that neural-crest-derived melanocyte development depends critically on Mitf-M.
RESULTS
Pigmentation phenotype of the Mitfmi-bw allele
Mitfmi-bw is a recessive mutation and when homozygous produces a white coat but normal sized eyes which remain black (Fig. 1A and B). This white coat colour results from the lack of melanocytes in the hair follicles (Fig. 1C and D). As in other white-spotted mutants, the choroid layer of the eye is also devoid of melanocytes (Fig. 1E and F). In addition, a previous histochemical examination of the inner ear has demonstrated the absence of intermediate cells or melanocytes in the stria vascularis (29). These observations suggest that Mitfmi-bw eliminates neural-crest-derived melanocytes from all parts of the body. Importantly, the retina of a homozygous Mitfmi-bw/Mitfmi-bw mouse shows its normal cellular arrangement with fully pigmented RPE cells in the outer-most layer (Fig. 1E and F) whilst most of the Mitf alleles cause abnormal RPE and/or retinal degeneration. The phenotype is the same even on a different genetic background, such as the original C3H, C57BL/6 and MSM derived from Japanese wild mice, Mus musculus molossinus (31).
Figure 1. Pigmentation defect of the Mitfmi-bw allele. Phenotypes of the wild-type (A, C and E) and Mitfmi-bw (B, D and F) alleles on the C3H background. (A) A control wild-type mouse. (B) A Mitfmi-bw/Mitfmi-bw homozygote showing white coat and normal sized black eyes. Paraffin sections of hair follicles (C and D) and retinae (E and F). Fully melanized dendritic melanocytes are visible in a hair follicle (C) and in the choroid layer (E) of wild-type mouse, but absent from Mitfmi-bw/Mitfmi-bw tissues (D and F). Note the presence of normal RPE both in wild-type (E) and in Mitfmi-bw/Mitfmi-bw (F) eyes. The homozygote (B) also lacks melanocyte in the inner ear and is completely deaf (29). ch, choroid layer; mc, melanocytes; pe, pigmented epithlium; pr, photoreceptor layer; on, outer nuclear layer; in, inner nuclear layer.
Molecular lesion of the Mitfmi-bw allele
The Mitf gene is composed of at least 12 coding exons and is transcribed from three different promoters, resulting in the production of three different isoform proteins, Mitf-M, Mitf-H and Mitf-A (10,23,30). Mitf-M (419 amino acids) was the first reported Mitf isoform (10). It is specifically expressed in melanocyte lineage cells and harbours at its N-terminus 11 amino acids encoded by exon 1M (30). Two other isoforms, RPE-enriched Mitf-A (30) (526 amino acids) and heart-type Mitf-H (23) (510 amino acids), accommodate in their N-termini 118 (Mitf-A) and 102 (Mitf-H) amino acids that originate from differential use of exons (Mitf-A, exons 1A and B1b; Mitf-H, exons 1H and B1b) 5[prime] to exons 2-9 (Fig. 2C and D).
Figure 2. The structure of Mitfmi-bw and various Mitf cDNAs produced. (A) Southern blot analysis of genomic DNAs. BamHI-digested genomic DNAs from wild-type C57BL/6 (lane wt) and Mitfmi-bw/Mitfmi-bw (lane bw) mice were hybridized with a 32P-labelled fragment derivd from the melanocyte-specific exon 1 (exon 1M). A hybridization band detected from Mitfmi-bw/Mitfmi-bw DNA (~11.4 kb) is larger than that obtained from wild-type DNA (~5.5 kb). Positions of size markers (in kb) are indicated on the left. (B) PCR amplification of intron 3. A primer set F4-R4 designed to amplify sequences of parts of intron 3 (420 bp) and exon 4 (40 bp) was used. The amplified band from wild-type DNA is 460 bp (lane wt) while that obtained from Mitfmi-bw/Mitfmi-bw DNA is ~7.7 kb (lane bw). Positions of size markers (in kb) are indicated on the left. (C) Schematic representation of a part of the Mitfmi-bw gene, showing the position of the L1 element insertion in intron 3 with nucleotide sequences of the L1-intron 3 junctions. Intronic sequences are in lowercase and the L1 sequences are in uppercase. The transcriptional orientation of the L1 insertion is indicated by an arrow. Two identical 15 bp sequences (underlined) flanking the L1 insertion indicate the target sequence duplication. Locations and orientations of PCR primers are depicted by arrows. Positions of four BamHI sites, two located in the L1 insertion close to the 3[prime]-end, and of the fragment (probe 1M) used as a hybridization probe in (A) are also indicated. Note that the exons and introns are not drawn to scale. Also, relative positions of exons 1A and 1H are not experimentally verified. The nucleotide sequences of Mitf intron 3 and L1bw have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession nos AB018704 and AB018705, respectively. (D and E) Structure of cDNAs for various Mitf isoforms. Three full-length Mitf isoforms, Mitf-H, Mitf-A and Mitf-M (D), and shorter variant Mitf proteins with an internal deletion, Mitfbw-H and Mitfbw-A (E), are produced by differential splicing of exons. Locations and orientations of primers for RT-PCR analyses are depicted by arrows and coding regions (grey box) and 5[prime]-UTRs (open box) are also indicated. Regions relevant to Mitf isoform variation are shown.
To determine the structural change in the Mitfmi-bw gene, we performed Southern blot analyses using probes corresponding to single or multiple exons of the wild-type Mitf gene. When the melanocyte-specific exon 1M fragment (probe 1M; Fig. 2C) was used as a probe, genomic DNA prepared from a homozygous Mitfmi-bw/Mitfmi-bw mouse gave larger hybridization bands compared with those obtained from wild-type DNA with several restriction enzymes (data not shown). For example, the mutant hybridization band was ~7 kb longer than the control fragment in BamHI digest (Fig. 2A). Since all the coding sequences of the Mi-fmi-bw allele were intact (see below), these results suggested that Mitfmi-bw is caused either by a small scale recombination or by an insertion into one of the introns. Therefore, we amplified introns close to exon 1M by PCR to examine possible alterations in their structures. A primer set F4-R4 (Fig. 2C), which would amplify a sequence comprising parts of intron 3 (420 bp) and exon 4 (40 bp), gave a PCR band of >7 kb from Mitfmi-bw/Mitfmi-bw DNA (Fig. 2B). Subsequently, cloning and sequencing of the entire 7.7 kb PCR product identified an insertion of a 7.2 kb sequence within Mitfmi-bw intron 3 with the integration site located 75 bp 5[prime] of exon 4. The insertion is flanked by 15 bp of the target sequence at both ends (Fig. 2C). A database search identified the inserted sequence as a LINE or L1 retrotransposable element. The L1 element (L1bw) exhibits strikingly high homology (>99%) with a recently identified, actively retrotransposing L1 subfamily termed the TF subfamily (32). No other alterations of sequences around the exon-intron boundaries or base changes in the coding sequences were found, despite extensive PCR and sequence analyses. Taken together with the fact that Mitfmi-bw arose spontaneously in the C3H line (25), we reasoned that this intronic insertion of L1bw is causal for the Mitfmi-bw mutation.
Effect of Mitfmi-bw on the expression of Mitf-A and Mitf-H
Because insertions of L1 elements have been shown to be associated with aberrant splicing of transcripts from inserted genes (33,34), we examined if this is the case in the Mitfmi-bw gene by cloning and sequencing multiple Mitf cDNAs isolated by RT-PCR. Three different sense PCR primers (FM, FA and FH) were used to distinguish transcripts for three Mitf isoforms, Mitf-M, Mitf-A and Mitf-H (Fig. 2C and D). As sources of poly(A)+ RNAs, we used skin and whole eyes, which contain both RPE and choroidal melanocytes, derived from 10-day-old mice.
When a sense primer specific to exon 1H (FH) was used with an antisense primer in exon 5 (R5), multiple bands were obtained from cDNA samples prepared from both wild-type and Mitfmi-bw/Mitfmi-bw tissues (Fig. 3A). Sequencing of cloned PCR fragments confirmed that each band represents one of the Mitf-H mRNA species produced by the differential splicing of exons located between exons 1H and 5, including exon B1b. To estimate the relative expression level of these differentially spliced mRNAs, we performed Southern hybridization with a Mitf-H-specific cDNA probe on samples that were normalized against the intensity of control [beta]-actin (Fig. 3F) hybridization bands. Quantitation of the data generated revealed that, overall, the entire Mitf-H cDNA hybridization signals detected in Mitfmi-bw/Mitfmi-bw samples were less intense than those detected in wild-type samples; ~40% reduction in the eye and 10% reduction in the skin, respectively (Fig. 3B). In addition, in the mutant mice a proportion of one spliced form (355 bp) that lacks a sequence corresponding to exons 2-4 was increased almost 3-fold in the eye and 2-fold in the skin, while that of the full-length form (763 bp) in both tissues was apparently reduced.
Figure 3. RT-PCR analyses of the expression of mRNA for various Mitf isoforms. Poly(A)+ RNAs were prepared from the skin and whole eyes of 10-day-old wild-type C57BL/6 (wt), Mitfmi-bw/Mitfmi-bw (bw) and KitW-v/KitW (Wv) mice. The RNA samples were reverse transcribed and subjected to PCR with primer sets specific for Mitf-H (A), Mitf-A (C), Mitf-M (E and G) or control [beta]-actin (F and G). To estimate the relative expression levels of differentially spliced Mitf mRNAs, standardized RT-PCR samples were probed with Mitf-H (B) or Mitf-A (D) cDNAs and the autoradiograms were quantitated. (A) An ethidium-stained gel, showing the major 763 bp fragments representing full-length Mitf-H mRNA and several shorter fragments representing differentially spliced mRNAs. Note the slight increase in a 355 bp fragment lacking sequences of exons 2-4 in Mitfmi-bw/Mitfmi-bw samples. (B) Relative quantification of differentially spliced mRNAs for Mitf-H. A reduction in the overall expression levels in Mitfmi-bw/Mitfmi-bw samples is evident. Note the moderate increase in a proportion of Mitf-H mRNAs lacking sequences of exons 2-4 in Mitfmi-bw/Mitfmi-bw samples. Various spliced forms are grouped into the following three types: full-length Mitf-H form; Mitfbw-H type lacking sequences of exon 2-4; other types. A relative amount (normalized to 100% for the amount of full-length form in wild-type skin) of each spliced form is given. Mean value from two independent experiments (two different RT-PCR tubes from the same RNA samples) is shown with bars indicating the higher values. (C) An ethidium-stained gel, showing 915 bp fragments representing full-length Mitf-A mRNA and multiple shorter fragments representing differentially spliced mRNA. Note the significant increase in 355 bp fragments lacking sequences of exons 2-4 in Mitfmi-bw/Mitfmi-bw samples. (D) Relative quantification of differentially spliced mRNAs for Mitf-A. A reduction in the overall expression levels of Mitfmi-bw/Mitfmi-bw samples is evident. Note the significant increase in a proportion of Mitf-A mRNAs lacking sequences of exons 2-4 in Mitfmi-bw/Mitfmi-bw samples. Various spliced forms are grouped into the following three types: full-length Mitf-A form; Mitfbw-A type lacking sequences of exon 2-4; other types. A relative amount (normalized to 100% for the amount of full-length form in wild-type skin) of each spliced form is given. Mean values from two independent experiments (two different RT-PCR tubes from the same RNA samples) are shown with bars indicating the higher values.(E) An ethidium-stained gel, showing the major 514 bp fragment representing full-length Mitf-M mRNA only in wild-type samples. Note the absence of differentially spliced Mitf-M transcripts both in wild-type and Mitfmi-bw/Mitfmi-bw samples. (F) An ethidium-stained gel, showing control [beta]-actin bands. (G) RT-PCR analysis of Mitf-M mRNA expression in another complete black-eyed white KitW-v/KitW mouse. Full-length 514 bp Mitf-M mRNA is not detected in 10-day-old KitW-v/KitW tissues (upper panel). Control [beta]-actin bands are shown in the lower panel.
Similarly, PCR amplification using a sense primer positioned in exon 1A (FA) with the R5 primer generated multiple bands corresponding to differentially spliced Mitf-A mRNAs derived from differential use of exons located between exons 1A and 5 in both genotypes (Fig. 3C). Relative quantitation of these bands indicated that as a whole Mitf-A mRNA expression is also impaired in Mitfmi-bw/Mitfmi-bw tissues, reduced by approximately two-thirds in both the eye and skin (Fig. 3D). As in the case of Mitf-H mRNAs, a proportion of the 507 bp fragments that represents mRNA produced by skipping of exons 2-4 was increased nearly 7-fold in the eye and >10-fold in the skin. In contrast, a proportion of 915 bp fragments representing the full-length Mitf-A mRNA was significantly reduced. The splicing pattern of exons 3[prime] to exon 5 was not altered in Mitfmi-bw/Mitfmi-bw tissues (data not shown).
The presence of Mitf-H and Mitf-A mRNAs in skin derived from Mitfmi-bw mice, which is devoid of neural-crest-derived melanocytes, indicates that these isoforms are expressed not only in the RPE but also in other non-pigment cells; it has been shown previously, for example, that although MITF-A mRNA is enriched in human cultured RPE cells, its expression is also detectable in a variety of tissues and cell lines at low levels (30). It is possible that the reduced levels of full-length Mitf-A and Mitf-H mRNAs in the skin and eye means that expression is not significantly altered in the non-melanocyte cell types and that the overall reduced level of expression reflects loss of the melanocyte population in the samples used. Whatever the reason for the reduced expression of the overall amount of Mitf-H and Mitf-A mRNAs is, it is nevertheless clear that the splicing pattern is disturbed by the L1bw insertion. Remarkably, the skipping of exons 2-4 in the transcripts for Mitf-H and Mitf-A do not result in translational frameshifts or premature termination codons and the shorter Mitf-H and Mitf-A mRNAs are still capable of encoding variant proteins with an internal deletion, Mitfbw-H (374 amino acids) and Mitfbw-A (390 amino acids) (Fig. 2E).
Functional analysis of the Mitf-H and Mitf-A variants
The results presented above prompted us to examine the function of shorter Mitf variants, Mitfbw-A and Mitfbw-H, as transcription factors, because if these variants are still capable of activating Mitf target genes in Mitfmi-bw RPE, any reduction in the functional levels of Mitf-A and Mitf-H would be compensated for by the presence of these variants, which would be derived from mRNAs present in substantially increased amounts. To answer this question, plasmids expressing various Mitf isoforms and their variants were transiently transfected into a melanocyte cell line, melan-c, with a reporter comprising 0.27 kb of the mouse Tyr gene promoter (35) fused to the luciferase gene. There are two Mitf-binding CATGTG motifs in the promoter. As shown in Figure 4, full-length Mitf-A and Mitf-H proteins activated reporter gene expression as efficiently as the control Mitf-M in melanocytes, but the deletion within the Mitfbw-A and Mitfbw-H variants completely abolished their ability to transactivate. The results obtained using fibroblasts were essentially very similar (data not shown). The inability of these Mitf variants to activate transcription is consistent with the fact that they lack exon 4, which encodes the major transcription activation domain of Mitf-M (36). Moreover, Mitfbw-A and Mitfbw-H variants might well act in a dominant-negative fashion (23,37), either by heterodimerizing with fully functional, full-length Mitf-A and Mitf-H proteins or by competing with the full-length proteins for binding to Mitf target sites; as such, the observed levels of mRNAs for Mitf-A and Mitf-H isoforms in Mitfmi-bw eyes might provide an overestimate of the amount of functional Mitf proteins present in the RPE.
Figure 4. Functional analysis of Mitf-A and Mitf-H variants. Transactivation capacity of various Mitf isoforms and their shorter variants on a luciferase reporter driven by the mouse Tyr gene promoter in melan-c. Full-length Mitf-A and Mitf-H proteins transactivate reporter gene expression as efficiently as the positive control Mitf-M does, whereas two shorter variant proteins, Mitfbw-A and Mitfbw-H, both of which are likely to be up-regulated in Mitfmi-bw/Mitfmi-bw tissues, are unable to transactivate reporter expression. The activity of each Mitf isoform is given relative to that obtained from the control pRc/CMV vector (CMV). Mean fold activation from three independent experiments is shown with SD. The reporter plasmid pMTL1, containing two Mitf-binding CATGTG motifs, has been described by Yasumoto et al. (35).
In developing mouse eyes, Mitf transcripts containing exon B1b, which is present in mRNAs for both Mitf-A and Mitf-H, are strongly expressed in presumptive RPE cells (30). However, in situ hybridization with shorter probes specific either for exon 1A (230 bases) or to 1H (91 bases) failed to give clear hybridization signals (data not shown). Amae et al. (30) reported that mRNA for MITF-A, a human homologue of Mitf-A, represents 95% of total MITF mRNAs in cultured human RPE cells, while MITF-M and MITF-A mRNAs represent 75 and 20% of total MITF isoform mRNAs, respectively, in human dermal melanocytes. MITF-H mRNA, on the other hand, is hardly detectable in both cell types. It is likely that Mitf isoforms with domain B1b (possibly Mitf-A and Mitf-H) support the development of pigmented Mitfmi-bw/Mitfmi-bw RPE cells even if the amount of the functional, full-length form of these isoforms were reduced in the cells.
Effect of Mitfmi-bw on the expression of Mitf-M
In 10-day-old mice, mRNAs for Mitf-M, the melanocyte-specific isoform, were amplified using a sense primer specific for exon 1M (FM) and the R5 primer (Fig. 2D) from wild-type skin and whole eyes containing RPE and choroidal melanocytes, but not from Mitfmi-bw/Mitfmi-bw tissues (Fig. 3E). Mitf-M mRNA was also not detected in the skin and whole eyes of KitW-v/KitW mice (Fig. 3G), another complete black-eyed mutant, confirming that the lack of Mitf-M mRNA in Mitfmi-bw/Mitfmi-bw mice is due to the absence of neural-crest-derived melanocytes. The result is in accordance with the observation that MITF-M mRNA is exclusively expressed in melanocyte lineage cells and is not detectable even in cultured human RPE cells (30).
We next asked whether the intronic insertion of L1bw affects the splicing of Mitf-M mRNA in any way or whether it exerts other effects and disturbs Mitf-M mRNA expression. Because melanocytes which specifically express Mitf-M mRNA are absent in Mitfmi-bw/Mitfmi-bw, one way of looking at the molecular changes would be to follow the developmental expression of Mitf-M in the abnormal or `dying' Mitfmi-bw/Mitfmi-bw neural-crest-derived cells. To this end we chose to examine transcripts from the Mitfmi-bw allele in normal pigmented melanocytes of heterozygous Mitfmi-bw/+ mice, hence in the presence of the wild-type allele. This allowed us to monitor the potential of the Mitfmi-bw allele to express Mitf transcripts in a cellular environment that does not interfere with the expression or function of the wild-type allele. The results revealed that differentially spliced Mitf-M mRNAs, such as those derived from skipping of exons 2-4, are not detectable in 10-day-old Mitfmi-bw/+ skin (Fig. 5A). More importantly, the relative amount of Mitf-M mRNA in the Mitfmi-bw/+ sample is reduced by ~70% compared with that detected in the wild-type sample (Fig. 5B). There may also be a slight reduction in the number of melanocytes in Mitfmi-bw/+ tissue (our unpublished observation). However, because it is unlikely that the integrated L1bw sequence disturbs Mitf mRNA expression from the wild-type allele (9,30), this 70% reduction should mainly reflect a very significant reduction in the amount of Mitf-M mRNAs or a complete block to Mitf-M mRNA expression from the Mitfmi-bw allele in individual Mitfmi-bw/+ melanocytes.
Figure 5. RT-PCR analyses of Mitf-M mRNA expression in Mitfmi-bw/+ mice. Poly(A)+ RNAs from the skin (skin) of 10-day-old wild-type (wt), Mitfmi-bw/+ (bw/+) and Mitfmi-bw/Mitfmi-bw (bw/bw) mice were reverse transcribed and subjected to PCR with primer sets specific to Mitf-M (upper panel) or control [beta]-actin (lower panel). (A) An ethidium-stained gel, showing the presence of much reduced, full-length 514 bp Mitf-M mRNA in 10-day-old Mitfmi-bw/+ mouse skin. Note the absence of differentially spliced Mitf-M transcripts in all samples. (B) Relative quantification of Mitf-M mRNAs expressed in Mitfmi-bw mutant mice. To estimate the relative amount of Mitf mRNA, standardized RT-PCR samples were probed with Mitf-M cDNA and the autoradiograms were quantitated. A relative amount (normalized to 100% for the amount in wild-type skin) of full-length Mitf-M mRNA present in each sample is given. Mean values from two independent experiments (two different RT-PCR tubes from the same RNA samples) are shown with bars indicating the higher values.
Functional analysis of the effect of Mitf intron 3 on Mitf-M expression
The reduction in Mitf-M mRNA expression in the Mitfmi-bw/+ background raised the possibility that the L1bw intronic insertion disrupts a sequence involved in Mitf-M mRNA transcription. As we have shown above, intact Mitf-M mRNAs seem not to be produced from the Mitfmi-bw allele. When the sequences of mouse Mitf intron 3 and human MITF intron 3 were compared, they showed exceptionally high homology over their entire length. Our preliminary experiments, in which the mouse Mitf intron 3 sequence was introduced into a reporter comprising the human melanocyte-specific MITF promoter fused to the luciferase gene and transiently transfected into melan-c cells, failed to detect any effect of the intronic sequence on promoter activity. Because there is a sequence similar to the cAMP-responsive element (CRE) close to the L1bw integration site, we also assessed whether or not intron 3 had any effect on the responseiveness of the melanocyte-specific MITF promoter to elevated cAMP levels. However, forskolin, a cAMP elevating agent, up-regulates reporter gene expression with or without the intronic sequence, indicating that the effect of cAMP is not affected by the intron (data not shown). Intron 3 is unlikely to contain cis elements primarily essential for Mitf-M mRNA expression, though it is entirely possible that the role of such elements would only be revealed in the context of the intact Mitf gene. Taken together, the results suggest that the obstruction of Mitf-M expression in melanocyte lineage cells is the most significant change caused by the L1bw insertion relevant to the Mitfmi-bw phenotype.
DISCUSSION
Here we describe the molecular defect underlying the Mitfmi-bw phenotype. Structural analyses of the Mitfmi-bw gene identified an insertion of a 7.2 kb novel L1 element, L1bw, in intron 3. The L1bw insertion exerts different effects on the expression of three known Mitf isoforms: a reduction in the overall expression levels of Mitf-A and Mitf-H mRNAs; the frequent skipping of exons 2-4 from pre-mRNAs for Mitf-A and Mitf-H; and most importantly, an apparent obstruction of Mitf-M mRNA expression. Changes in the expression patterns of Mitf-A and Mitf-H mRNAs might lead to a reduction in the amount of functional, full-length Mitf-A and Mitf-H isoforms in RPE lineage cells. In contrast, the alteration in Mitf-M mRNA expression should result in the elimination of functional melanocyte-specific Mitf-M. It seems likely that the presence of full-length Mitf-A and Mitf-H are sufficient to support normal RPE development while the absence of Mitf-M is responsible for the loss of melanocytes in Mitfmi-bw/Mitfmi-bw mice. The results suggest that melanocyte development depends critically on a single Mitf isoform, Mitf-M, and that auditory-pigmentary symptoms could result from a specific mutation affecting the production of functional Mitf-M in mice.
Effects of multiple promoters and L1bw insertion on Mitf isoform expression
RT-PCR analyses of Mitf-M mRNA expression in Mitfmi-bw homozygous and heterozygous mice revealed that L1bw insertion in Mitfmi-bw obstructs production of Mitf-M mRNAs. This is unexpected because only partial inhibition of mRNA transcription was detected in two other Mitf isoform mRNAs and in the case of the spastic gene (34,38). How the L1bw insertion specifically obstructs Mitf-M mRNA expression is not clear, but there are three potential explanations. (i) Aberrantly spliced Mitf-M transcripts are extremely unstable and rapidly degraded; destabilization of MITF mRNA has been observed previously for both mouse Mitfmi-ce and Syrian hamster anophthalmic white mutations (23,39). (ii) The L1bw insertion specifically induces premature termination of Mitf-M mRNAs and produces truncated transcripts. It has been shown that the intronic insertion of an active LINE element, the I factor, in the Drosophila white gene causes aberrant pre-mRNA maturation depending on its position and orientation (40). (iii) The L1bw insertion specifically disrupts sequences involved in Mitf-M mRNA production, which would imply an interplay between the splicing machinery and transcription factors that bind to the melanocyte-specific promoter driving Mitf-M expression. The analysis of Mitf-M transcripts in mice heterozygous for the Mitfmi-ws allele, a deletion mutation in a region encompassing intron 3 (23), will be informative in deciding if this is the case. However, although it is clear that the Mitfmi-bwphenotype results from insertion of the L1 element into intron 3, which of the possible explanations outlined above accounts for the specific loss of the MITF-M isoform remains to be established.
Implications for human WS2 conditions
Previously, screening for MITF mutations in patients with WS2 or WS2-related conditions has been carried out using primers designed to detect base changes in the coding region and the flanking splice donor/acceptor sites (6,16-20). This is partly because sequences potentially involved in the expression (transcription and pre-mRNA splicing) of MITF, which is predicted to span ~200 kb (41), were not known. In two WS2 families, a MITF mutation was identified in the GT donor splice site at the end of exon 1M and the mutation was predicted to cause translational read-through to a termination codon in intron 1 (6,17). While this mutation should abrogate the production of melanocyte-specific MITF-M, it might not seriously disturb the expression of other MITF isoforms, MITF-A and MITF-H. Although biochemical evidence is clearly needed, these cases could represent WS2 resulting from a MITF-M-specific mutation in the heterozygous state.
Our analysis of the Mitfmi-bw mutation suggests that WS2 might also arise as a result of insertions into intron 3, and perhaps other introns, which may specifically affect expression of the melanocyte-specific isoform of Mitf, Mitf-M. Indeed, compound heterozygotes between the Mitfmi-bw allele and other recessive Mitf alleles may result in an enhanced reduction in the functional level of Mitf-M protein specifically in melanocytes, thereby creating mice with aural conditions similar to WS2 patients (39). In addition, it has recently been shown that the 5[prime]-flanking sequence of MITF exon 1M is able to direct melanocyte-specific transcription in cultured cells (42) and that PAX3, associated with WS1/3, is one of the transcription factors that can bind and transactivate the promoter (43). Given our results showing that melanocyte development is critically dependent on MITF-M, sequence alterations in the melanocyte-specific promoter, including the PAX3-binding site located between positions -263 and -238, might also cause melanocyte deficiency through a reduction in the amount of functional MITF-M. Thus, screening for coding sequence or splice site mutations alone may under-estimate the contribution of mutations in the MITF gene to WS2 and other mutations which specifically affect the MITF-M isoform may also be identified by screening the melanocyte-specific promoter and introns.
The role of Mitf in the development of melanocytes and RPE
The differential effect of Mitfmi-bw on neural-crest-derived melanocytes and optic cup-derived RPE may imply that different Mitf isoforms are required for the establishment of the two different types of pigment cells with distinct embryonic origins. However, it should be borne in mind that the phenotypes of WS2 patients and Mitf mutant mice suggest that neural-crest-derived melanocytes are significantly more sensitive to reduced levels of functional Mitf compared with the RPE. As such, it is also possible that the apparent differential requirement for the various Mitf isoforms in Mitfmi-bw mice may simply reflect a combination of increased sensitivity and greater reduction in functional Mitf levels in the neural-crest-derived population of pigment cells.
Comparison of the phenotype and molecular defect of Mitfmi-bw with those of other hitherto described Mitf alleles (9,22-24,39,44), particularly of Mitfmi-ws mentioned above, should provide further insights into the function of different Mitf isoforms in the development of pigment cells. When homozygous, Mitfmi-ws produces a complete white coat and near normal sized pink eyes without any other abnormalities (10,22,23). It has been shown that shorter Mitf mRNAs, probably corresponding to Mitf-A and Mitf-H without a region comprising exons 2-4, are produced at approximately wild-type levels in Mitfmi-ws hearts (23). As such, the lack of melanocytes in Mitfmi-ws mice signifies the importance of the major transcription activation domain encoded by exon 4 (36) in melanocyte development. The activation domain, which is predicted to adopt an [alpha]-helical conformation, is present in all three Mitf isoforms. So how can the presence of normal sized pink eyes or unpigmented but otherwise normal RPE cells of Mitfmi-ws mice be explained? Since neither the Mitfbw-A nor Mitfbw-H variants were able to activate transcription from the Mitf target Tyr gene promoter (Fig. 4), regions outside the helical activation domain may be critical for the transcriptional activation of genes involved in normal RPE development and to prevent hyperproliferation of cells comprising the outer layer of the optic cup (44) and support development of non-pigment cell types, despite the fact that the ability to activate transcription from the pigmentation genes appears to be defective in Mitfmi-ws RPE cells. In this respect, it is significant that three Mitf isoforms possess different transactivation potentials on the Tyr promoter. Long N-terminal domains of Mitf-A and Mitf-H may interact with as yet unidentified or RPE-specific transcription cofactors, as the helical activation domain recruits the CBP/p300 transcription co-activator (36,45). It is equally possible that heterodimers with other bHLH-LZ factors substitute for the function of the Mitf homodimer in Mitfmi-ws RPE.
The lack of any Mitf-M transcripts in two independent black-eyed white mice (Fig. 3) confirmed that only neural-crest-derived melanocytes express this particular Mitf isoform in vivo, as has been anticipated from the expression pattern of the human homologue, MITF-M (30). However, at present we cannot rule out the possibility that MITF-A is also involved in melanocyte development at particular developmental stages or under certain circumstances, considering that MITF-A mRNA is expressed at low levels almost ubiquitously and is present in melanocyte lineage cells as well (30). Observations that some Mitf compound heterozygotes, including MitfMi-wh/Mitfmi-bw and MitfMi-wh/Mitfmi-ws, produce pigmented or coloured spots (11) and the appearance of occasional black pigmented spots on the dorsum or on the rump of Mitfmi-bw homozygotes (our unpublished observation) may imply that this is the case. Establishment of melanocyte cell lines from these pigmented spots and subsequent characterization of the Mitf isoforms expressed in those cells will give a clearer picture.
L1 retrotransposition-induced mutations in humans and mice
In recent years, L1 retrotransposons have attracted much attention, not only because they are the predominant mobile element present in the mammalian genome and are the causal agents of several human diseases, but also because of their potential applications in medical and biological sciences (46). The association of L1 with human diseases was first recognized when L1s were found to disrupt the factor VIII gene in patients with haemophilia A (47). Insertional mutations by recently retrotransposed L1s are found in four genes, the factor VIII gene (46,48), the DMD gene (49,50), the APC gene (51) and the [beta]-globin gene (46). All but one of these human L1s belong to a transcriptionally active group, the Ta subset, while the TF subfamily appears to be the major retrotransposition-competent L1 in present day mice. Two of four previously characterized mouse spontaneous alleles associated with L1 retrotranspositions are caused by full-length members of the TF subfamily (46), L1spa in the spastic gene and L1Orl in the Orleans reeler gene, as described. Thus, the Mitfmi-bw allele caused by the L1bw insertion is only the third of such cases. Given that L1bw has sufficient 5[prime]-untranslated region (5[prime]-UTR) for retrotransposition (32) but no nonsense mutations in the open reading frames and also that the most closely related L1, Md-Tf5, is capable of retrotransposing autonomously at high frequency in cultured mouse cells (52), it is likely that L1bw, derived from a recent retrotransposition event into the Mitf gene, retains a capacity to retrotranspose in the mouse genome. Further characterization of L1bw, a new member of TF, will provide an opportunity to understand the TF subfamily in the mouse and the role of L1s in shaping the mammalian genome.
MATERIALS AND METHODS
Mice
The original mutant strain (C3HBSt) carrying the Mitfmi-bw allele was kindly provided by Dr Walter C. Quevedo Jr (Brown University, Providence, RI). The Mitfmi-bw allele was then backcrossed onto the C57BL/6 background (13 generations) and maintained in our laboratory. After several years, however, the Mitfmi-bw homozygous mice became difficult to breed and the size of the colony became dangerously small (<10). In our attempt to rescue the Mitfmi-bw allele, two male mutant mice (C57BL/6-Mitfmi-bw/Mitfmi-bw) crossed with C3H/He female mice gave offspring and now Mitfmi-bw is maintained on the mixed background (N3 generation on the C3H background). In this study, genomic DNA and RNAs were prepared from mice either on the C57BL/6 background (N12) or on the mixed backgrounds (N2 and N3 on the C3H background).
C57BL/6CrSlc, C3H/HeSlc and C57BL/6-KitW-v/KitW mice were purchased from Japan SLC (Hamamatsu, Japan) and maintained in our laboratory.
Cells and transfection assays
Melan-c mouse albino melanocytes (53) were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 200 ng/ml 12-O-tetradecanoylphorbol-13-acetate, 100 µM [beta]-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C under 10% CO2. BALB 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C under 5% CO2. Transfections were performed essentially as described (36) with the following modifications. The amount of lipofectamine used per µg of DNA was 7 µl for melan-c and 10 µl for BALB 3T3 cells. Transfected cells were harvested after 48-72 h and assayed for luciferase and [beta]-galactosidase activities. In some experiments, transfection medium, containing DNA-lipofectamine complex, was replaced with fresh medium supplemented with 20 µM forskolin. [beta]-Galactosidase activity of a co-transfected reporter was used as an internal control to correct for the variability in transfection efficiency.
Genomic DNA isolation and PCR analysis
Preparation of genomic DNA from the liver and Southern hybridization were carried out as described previously (54). Genomic DNA extracted from a C57BL/6 male was used as a control wild-type sample. PCR amplification was performed using recombinant Taq DNA polymerase (TaKaRa) or the Expand Long PCR System (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions with the following primer sets: F1 (sense primer in exon 1M, 5[prime]-TCGGGATGCCTTGTTTATGGTG-3[prime]) and R1 (antisense primer in exon 2, 5[prime]-CAGTTGGAGTTAAGAGTGAGCATAGCC-3[prime]); F2 (sense primer in exon 2, 5[prime]-CAGGTAAAGCAGTACCTTTCTACCACTTTAGCA-3[prime]) and R2 (antisense primer in exon 3, 5[prime]-GTTCATACCTGGGCACTCACTCTCT-3[prime]); F3 (sense primer in exon 3, 5[prime]-TTTTATAAGTTTGAGGAGCAGAGCAGG-3[prime]) and R4 (antisense primer in exon 4, 5[prime]-TCCATCAAGCCCAAAATTTCTTCATTATAACT-3[prime]); F4 (sense primer in intron 3, 5[prime]-GGAAAAGGGAAGTGGTAGCTTTGTG-3[prime]) and R3 (antisense primer in exon 4, 5[prime]-TTCCAGGCTGATGATGTCATCAATTACATC-3[prime]). The amplified products were electrophoresed through agarose gels, cloned into pCRII (Invitrogen, Carlsbad, CA) and sequenced.
RNA isolation and RT-PCR analysis
Total RNA samples were extracted from tissues derived from several mice by the acid guanidinium thiocyanate-phenol-chloroform extraction method (55). Poly(A)+ RNAs were selected using Oligotex-dT30 latex beads (Nihon Roche, Tokyo, Japan). Single-stranded cDNAs were synthesized from the poly(A)+ RNA samples with avian myeloblastocis virus reverse transcriptase (Seikagaku Kogyo, Tokyo, Japan) using oligo(dT)12-18 or R5 as primer and added directly to the PCR mixture. The primers used were FA (sense primer in exon 1A, 5[prime]-ATGCAGTCCGAATCGGGAATCGTGG-3[prime]), FH (sense primer in exon 1H, 5[prime]-GAACACCTTAAAGGAAGAAAGATGGAGGCGCTTAG-3[prime]), FM (sense primer in exon 1M, 5[prime]-TGGTCTGCGGTGTCTCCTGGG-3[prime]) and R5 (antisense primer in exon 5, 5[prime]-TGTGGGGGAAAATACACGCTGTGAGC-3[prime]).
To quantify the relative amounts of differentially spliced Mitf transcripts, different amounts of single-stranded cDNA samples synthesized from the same poly(A)+ RNA sources were subjected to 20 cycles of PCR in each experiment. The RT-PCR experiments were repeated using the same poly(A)+ RNA samples. The amplified products were resolved on agarose gels, blotted onto Hybond-N+ nylon membranes (Amersham, Amersham Place, UK), hybridized with 32P-labelled Mitf cDNA probes and the autoradiograms were quantitated using a bio-image analyser and Molecular Imager system (Bio-Rad, Hercules, CA). The linearity between the amount of PCR products and that of the single-stranded cDNA input into the reaction mixture was ascertained in each experiment. To standardize the amount of total cDNA input, the cDNA for [beta]-actin was also amplified and quantitated as described above.
Plasmid clones
Complementary DNAs for Mitf-A and Mitf-H (30) were isolated by the RACE method using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA) from a transformed mouse melanocyte (TM10) cDNA library (56). A cDNA clone for Mitf-M, pBluescript-Mitf-M (pBS-Mitf-M), has been described previously (57). The full-length Mitf-M cDNA lacks an alternative six amino acid insertion (ACIFPT) (10,23) and is flanked by EcoRI and BglII restriction sites at both ends.
To construct pRc/CMV-Mitf-M, the full-length Mitf-M cDNA was excised by digesting at the flanking XbaI and ApaI restriction sites and cloned directly into the XbaI and ApaI sites of the pRc/CMV expression vector (Invitrogen). For construction of pRc/CMV-Mitf-A and Mitf-H, the 5[prime] portions of Mitf-A and Mitf-H cDNAs were amplified by PCR with specific primer sets, FA-R5N (new antisense primer in exon 5, 5[prime]-GGTCGATCAAGTTTCCAGAGACG) and FH-R5N, respectively, and cloned into pCRII and sequenced. Subsequently, sequences 5[prime] to the internal BamHI site of Mitf-A and Mitf-H cDNAs were excised as NotI-BamHI fragments and ligated to pBS-Mitf-M digested with NotI and BamHI to make full-length Mitf-A and Mitf-H cDNA clones (pBS-Mitf-A and pBS-Mitf-H). The full-length Mitf-A and Mitf-H cDNAs were digested at the flanking EcoRI sites and recloned into the EcoRI site of pBluescript II SK+ (pBSII; Stratagene, La Jolla, CA). Finally, the full-length Mitf-A and Mitf-H cDNAs were excised and cloned into the XbaI and ApaI sites of pRc/CMV. For construction of pRc/CMV-Mitfbw-A and Mitfbw-H, 5[prime] portions of Mitfbw-A and Mitfbw-H cDNAs were amplified by RT-PCR with primer sets FA-R5 and FH-R5, respectively, cloned into pCRII, sequenced, and sequences 5[prime] to the internal DraIII site of Mitf-A and Mitf-H cDNAs were excised as EcoRI-DraIII fragments. The 5[prime] EcoRI-DraIII fragments were cloned with a 3[prime] DraIII-EcoRI fragment derived from pBS-Mitf-M into the EcoRI site of pBSII to make full-length Mitfbw-A and Mitfbw-H cDNAs (pBSII-Mitfbw-A and pBSII-Mitfbw-H). Then, the full-length Mitfbw-A and Mitfbw-H cDNAs were excised and cloned into the XbaI and ApaI sites of pRc/CMV.
ACKNOWLEDGEMENTS
We wish to thank Dr L. Lamoreux for valuable discussions and kind advice on how to save our Mitfmi-bw mutant mouse strain from extinction, and Dr T. Shiroishi for supplying the MSM mouse strain established from Japanese wild mice in his laboratory and also for rescuing the Mitfmi-bw mice. We are also grateful to Dr D. Bennett for melan-c cells, Dr N. Yanai for BALB 3T3 cells and Dr K. Sogawa for technical advice. This study was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (to H.Y.).
REFERENCES
*These authors contributed equally to this work
+Present address: Department of Biology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan
§To whom correspondence should be addressed. Tel: +81 22 217 6692; Fax: +81 22 263 9206; Email: hyamamot{at}mail.cc.tohoku.ac.jp
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