Interallelic Complementation at the Mouse Mitf Locus



Interallelic Complementation at the Mouse

Mitf

Locus

Eirı´kur Steingrı´msson,*

_{Heinz Arnheiter,}

_{Jo´n Hallsteinn Hallsson,*}

_{M. Lynn Lamoreux,}

Neal G. Copeland

_{and Nancy A. Jenkins}

*Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavı´k, Iceland, †_{Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National}

Institutes of Health, Bethesda, Maryland 20892 and‡_{Mouse Cancer Genetics Program,} National Cancer Institute, NCI-FCRF, Frederick, Maryland 21702-1201

Manuscript received July 30, 2002 Accepted for publication October 15, 2002

ABSTRACT

Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types, including melanocytes, retinal pigment epithelial cells of the eye, and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The many mutations isolated at the locus have different effects on the phenotype and can be arranged in an allelic series in which the phenotypes range from near normal to white microphthalmic animals with osteopetrosis. Previous investigations have shown that certain combinations ofMitfalleles complement each other, resulting in a phenotype more normal than that of each homozygote alone. Here we analyze this interallelic complementation in detail and show that it is limited to one particular allele,MitfMi-white_(MitfMi-wh_{), a mutation affecting the DNA-binding domain. Both}

loss- and gain-of-function mutations are complemented, as are otherMitfmutations affecting the DNA-binding domain. Furthermore, this behavior is not restricted to particular cell types: Both eye development and coat color phenotypes are complemented. Our analysis suggests thatMitfMi-wh_{-associated interallelic}

complementation is due to the unique biochemical nature of this mutation.

M

pheno-(Mitf) affect the development of several different type (large belly spots and coat color dilution) and an

cell types. Common to all the mutations are defects in intermediate homozygous phenotype (white coat and

the neural-crest-derived melanocytes, resulting in reduc- partial microphthalmia). Thus, the relative effects of

tion or lack of pigmentation in the coat, eye, and inner these two mutations are different in the hetero- and

ear. Most of the mutations also affect pigmented epithe- homozygous conditions. Although many moreMitf

mu-lial cells of the eye and mast cells, while only a few tations have been isolated since, Gru¨ neberg’s

observa-mutations result in osteoclast defects. Approximately tions still hold true; the semidominant phenotype of

one-half of theMitfalleles are semidominantly inherited theMitfMi-wh_{mutation is the most severe phenotype}

asso-and show white spotting asso-and/or coat color dilution ciated with any mutation at the locus while its

homozy-when heterozygous with wild type. The remaining alleles _{gous phenotype is only intermediate.Konyukhovand}

are recessive and exhibit a phenotype only in homozy- _{Osipov(1968) analyzed the relationship between these}

gous condition. TheMitfmi-spotted_(Mitfmi-sp_{) mutation is}

un-two alleles further and showed that heteroallelic animals

usual in that it displays a visible phenotype only in com- _{show interallelic complementation with respect to}

ef-binations with other mutations at the locus (reviewed _{fects on the eye: Mitf}Mi-wh_/Mitfmi _{animals have normal}

byMoore1995). _{eye size while homozygous littermates exhibit severe}

In 1953, Gru¨ neberg noted the paradoxical relations _(Mitfmi_/Mitfmi_{) or intermediate (Mitf}Mi-wh_/MitfMi-wh₎

mi-of the semidominantMitfmicrophthalmia_(Mitfmi_{) andMitf}Mi-wh

crophthalmia. Similar analysis with a few otherMitf

al-mutations (Gru¨ neberg 1953). While theMitfmi

muta-leles also showed interallelic complementation. For

ex-tion exhibits a weak phenotype in the heterozygous _{ample, Schaible (1963) showed that a combination}

condition (occasional head blaze and belly streaks) and _{of theMitf}Mi-wh _{allele with the Mitf}mi-black-eyed white _(Mitfmi-bw₎

a very strong phenotype in the homozygous condition _{mutation resulted in white animals with pale yellow spots}

(microphthalmia, white coat, and osteopetrosis), the _{(on the C3HxB6 background they have pigmented spots}

on the back). Homozygotes for both mutations are com-pletely white. Similarly, heteroallelic combinations of

1_{Corresponding author:Department of Biochemistry and Molecular}

MitfMi-wh _{with Mitf}mi-white spot _(Mitfmi-ws_{) produced animals} Biology, Faculty of Medicine, University of Iceland, Vatnsmy´rarvegur

with dark eyes of normal size and a spotted, checker-16, 101 Reykjavı´k, Iceland. E-mail: [email protected]

2_{Present address:8255 Sandy Point Road, Bryan, TX 77807. board-like coat with yellowish-brown to gray colors}

MATERIALS AND METHODS

(Hollander1964). Homozygotes for theMitf

muta-tion are white with pink eyes. This suggests that interal- _{The followingMitfmutants were used in this study: C57BL/}

lelic complementation is a common phenomenon at 6J-MitfMi-wh_{, C57BL/6J-Mitf}mi-sp_{, C57BL/6J-Mitf}mi-red-eyed white_(Mitfmi-rw_),

C57BL/6J-Mitfmi-eyeless white_(Mitfmi-ew_{), C57BL/10-Mitf}mi-black and white spot

theMitflocus and that both eye and coat color defects

(Mitfmi-bws_{), C57BL/6J-Mitf}mi_{, 82UT-Mitf}Mi-oak ridge _(MitfMi-or_),

82UT-are affected. Although this phenomenon has never been

Mitfmi-brownish _(Mitfmi-b_{), and mixed [C3H/C57BL/6J]-Mitf}mi-vga-9

characterized systematically in detail, these observations

(Table 1). These strains are maintained at the National Cancer

suggest that interallelic complementation reveals an im- _{Institute in Frederick, Maryland, and at the National Institute}

portant aspect of the nature of theMitflocus. of Neurological Disorders and Stroke, National Institutes of

Health, in Bethesda, Maryland. The mice were mated

systemat-The MITF protein is a member of the MYC supergene

ically to generate the different allelic combinations. At least

family of basic-helix-loop-helix-leucine zipper

(bHLH-three different independent crosses were set up for generating

Zip) transcription factors and is most closely related to _{each combination and multiple offspring (⬎25) were analyzed}

the TFE3, TFEB, and TFEC proteins (Hodgkinson et _{from each cross. The phenotypes of the resulting animals were}

al. 1993; Hugheset al. 1993). Like other members of visually inspected, the animals were photographed at 6 weeks

of age (or earlier), and eyes, skin, and Harderian gland were

the bHLH-Zip family, MITF has been shown to bind

dissected for histologic analysis. All tissue specimens were fixed

the CANNTG E-box sequencein vitroas either a

homo-in Bouhomo-ins fixative, sectioned, and then stahomo-ined with

hemotoxy-dimer or a heterohemotoxy-dimer with TFE3, TFEC, and TFEB _{lin and eosin. For the evaluation of melanin in Harderian}

(Hemesathet al. 1994). The basic domain is the DNA- _{glands and in the retinal pigment epithelium (RPE) of the}

binding domain of the protein while the HLH and Zip eye, tissues were stained with Fontana-Masson.

domains are responsible for dimerization. Consistent with its role as a regulator of gene expression, MITF is

RESULTS

primarily located in the nucleus (Takebayashi et al.

1996) where it can activate expression from pigment _{Heteroallelic combinations ofMitfmutations:To}

per-cell, mast per-cell, and osteoclast specific promoters (Bent- _{form a systematic study of theMitf-associated interallelic}

leyet al. 1994;Yasumotoet al. 1994;Moriiet al. 1996; _{complementation, availableMitfalleles were crossed to}

YasumotoandShibahara1997;Motyckovaet al. 2001). _{each other in all possible combinations. The phenotypes}

The molecular and biochemical defects associated _{of the resulting heteroallelic offspring were studied by}

with most of the Mitf alleles have been determined _{visual inspection of coat color and eye size. The alleles}

(Hodgkinsonet al. 1993;Hugheset al. 1993;Hemesath _{used in this study are described in Table 1 and range in}

et al. 1994; Steingrı´msson et al. 1994, 1996; Yajima _{phenotype from very mild (e.g.,Mitf}mi-sp_{) to severe (}

Mitfmi₎

et al. 1999; Hallssonet al. 2000). The semidominant _{and in mode of inheritance from recessive (Mitf}mi-sp_,

mutations characterized to date affect either the DNA- _Mitfmi-rw_,Mitfmi-ew_{, Mitf}mi-vga9_{) to semidominant (Mitf}Mi-wh_,

binding or the transcriptional activation domains of the _MitfMi-or_{, Mitf}Mi-b_{, Mitf}mi_{). In all the crosses made, the}

protein while dimerization domains are unaffected _{number of heteroallelic progeny was according to}

Men-(Hodgkinson et al. 1993; Steingrı´msson et al. 1994, _{delian ratios (data not shown) and the phenotypes were}

1996). The mutant proteins cannot bind DNA; however, _{consistent among the different progeny and litters of}

they can still dimerize with proteins such as TFE3 and _{each cross.}

thereby interfere with DNA binding of the partner _{The phenotypes of the resulting combinations are}

(Hemesathet al. 1994;Steingrı´mssonet al. 1996). The _{described in Table 2 and in Figures 1–3. In most cases}

dominant-negative behavior of these mutant proteins _{where the homozygous phenotypes of the two alleles}

in vitrothus accounts for the phenotype seen in heterozy- _{are similar to each other, the phenotypes of the}

hetero-gous mice. Consistent with this, the recessive mutations _{allelic combinations are the same as each of the}

homozy-affect either the dimerization domain of the MITF pro- _{gotes. For example, Mitf}Mi-or_/Mitfmi _{animals are white}

tein or the transcription of theMitfgene, resulting in _{and microphthalmic and have severe osteopetrosis just}

little or no MITF production (Hodgkinsonet al. 1993; _{like the respective homozygotes (Mitf}Mi-or_/MitfMi-or _and

Hugheset al. 1993;Steingrı´mssonet al. 1994;Yajima _Mitfmi_/Mitfmi_{; Table 2). Similarly,Mitf}mi-ew_/Mitfmi-vga9

ani-et al. 1999). _{mals have a white coat and severe microphthalmia but no}

Despite the detailed molecular and biochemical anal- osteopetrosis, just like theMitfmi-ew_/Mitfmi-ew_andMitfmi-vga9_/

ysis of the Mitfmutations, no satisfactory explanation Mitfmi-vga9_{homozygotes (Table 2). In most cases in which}

for the interallelic complementation has emerged. Here, the homozygous phenotypes are different, however, the

we perform a detailed genetic analysis of the interallelic heteroallelic animals exhibit an intermediate

pheno-complementation at the Mitf locus and show that it type. For example,Mitfmi-ew_/

Mitfmi-sp_{animals have white}

is restricted to the MitfMi-wh_{mutation, a mutation with feet, head, and belly while the rest of the coat is gray}

unique characteristics. Our analysis suggests that the (Table 2 and Figure 1A). This phenotype is intermediate

nature of theMitf-associated interallelic complementation between the white microphthalmicMitfmi-ew_/Mitfmi-ew

(Ta-ble 2) and near normalMitfmi-sp_/Mitfmi-sp_{(Table 2, Figure}

Figure1.—Phenotypes resulting fromMitf mutations and heteroallelic combinations. (A)Mitfmi-sp_/Mitfmi-ew_{. (B)Mitf}mi-sp_/Mitfmi-sp_:

Note the apparently normal appearance of this animal. (C)MitfMi-b_/Mitfmi-rw_{: The pigmented spots are agouti due to the C3H}

background of the MitfMi-b _{mutation. (D) Mitf}mi-rw_/Mitfmi-rw_{: Note the black head spots. (E) Mitf}mi-ew_/Mitfmi_{: This animal lacks}

pigment and shows severe microphthalmia but no osteopetrosis. (F) MitfMi-wh_/Mitfmi-sp_{. (G) Mitf}mi-rw_/Mitfmi_{: Note the severe}

microphthalmia. (H)MitfMi-wh_/Mitfmi-rw_{: Note the faintly pigmented patch on their heads (dotted line).}

1B) animals. Similarly,MitfMi-b_/Mitfmi-rw_{animals are white white spots and normal-size eyes (WolfeandColeman}

1964; Table 2, Figure 1F). Although Mitfmi-sp

homozy-with large pigmented spots (Table 2 and Figure 1C),

a phenotype intermediate between the white MitfMi-b _{gotes have a normal appearance, a reduction in the}

activity of the tyrosinase enzyme has been detected in

homozygotes (Table 2) and Mitfmi-rw_/Mitfmi-rw _animals,

which are white with a pigmented spot on the head their skin (WolfeandColeman1964). All heteroallelic

combinations involvingMitfmi-sp_{show a more severe}

de-(Table 2, Figure 1D). In all these cases, one of the alleles

encodes a partially functional protein. Interestingly, the fect in pigmentation than these mutations show in

het-erozygous combinations with wild type, albeit less than

combination of Mitfmi-ew _andMitfmi_{results in white,}

se-verely microphthalmic animals (Figure 1E); this pheno- what is observed in homozygotes for the other allele

(Table 2). For example,Mitfmi-rw_/Mitfmi-sp_{animals have}

type is identical to that ofMitfmi-ew _{homozygotes while}

Mitfmi_{homozygous animals also exhibit osteopetrosis. a large belly spot (Table 2), a phenotype not seen in}

either Mitfmi-rw_/⫹ _{or Mitf}mi-sp_/Mitfmi-sp _{animals. Also,}

All combinations involving the Mitfmi-sp_{mutation fall}

into the intermediate group; the resulting phenotypes MitfMi-b_/Mitfmi-sp_{animals exhibit a diluted coat color}

phe-notype while MitfMi-or_/Mitfmi-sp _{animals are white with}

are in direct relation to the severity of the allele to which

Mitfmi-sp_{is crossed (Table 2). This mutation was originally spots that gradually lose their pigmentation with age}

(Table 2). This gradual depigmentation is similar to

found in a colony ofMitfMi-wh_{animals (Wolfeand}

Cole-man1964). While animals homozygous for theMitfmi-sp _{that observed for the Mitf}mi-vitilago _(Mitfmi-vit_{) mutation}

(Lerneret al. 1986;Lamoreuxet al. 1992). mutation cannot be distinguished from their wild-type

littermates,MitfMi-wh_/Mitfmi-sp_{heterozygotes are tan with It is interesting to compare the phenotypes ofMitf}mi-sp

Figure2.—Phenotypes asso-ciated with combinations with the Mitfmi-vga9

loss-of-func-tion mutaloss-of-func-tion. (A) Mitfmi-vga9_/ Mitfmi-vga9_{: Note the severe}

mi-crophthalmia. (B) Mitfmi-ew_/ Mitfmi-vga9_{. (C)Mitf}Mi-wh_/Mitfmi-vga9_:

Figure3.—Interallelic complementation at theMitflocus. (A)MitfMi-wh/⫹_{(pigmented, in the back) andMitf}Mi-wh_/MitfMi-wh_(white

and microphthalmic, in the front) animals: Note the intermediate microphthalmia. (B)Mitfmi_/Mitfmi_{: Note the severe microphthalmia}

and small size of the 3-week-old animals. (C)MitfMi-wh_/Mitfmi_{. Eyes are of normal size (arrows) although eye pigment is somewhat}

reduced compared to normal. (D)MitfMi-wh_/Mitfmi-ew_{. The eyes are of normal size (arrows). (E)Mitf}Mi-or_/MitfMi-or_{(front) andMitf}Mi-wh_/ MitfMi-or_{(back) animals. Note the normal eye size in the compound heterozygote (arrow). (F)Mitf}Mi-wh_/MitfMi-b_andMitfMi-b_/MitfMi-b

animals. The two compound heterozygotes (leftmost animal and the animal on top) show light-brown coat color while the two MitfMi-b_{homozygotes (bottom) are completely white. Neither genotype affects eye size. (G) Mitf}mi-bws_/Mitfmi-bws_{: Note the black}

spots on otherwise white background. Eye size is normal. (H)MitfMi-wh_/Mitfmi-bws_{. The phenotype is intermediate between each}

homozygote (compare to A and G) and no complementation is observed.

homo- and heterozygotes with the phenotypes of het- supported by genetic studies (Steingrı´msson et al.

2002; see below). eroallelic combinations involving the loss-of-function

Mitfmi--vga9_{and the recessiveMitf}mi-ew_{mutations. As already The Mitf}mi-rw _{mutation also has unique features.}

Ho-mozygotes for this mutation are white with pigmented

explained, no phenotype is visible in Mitfmi-sp_/

Mitfmi-sp

animals, suggesting that the mutant protein has some spots of somewhat variable size on the head (Figure

1D); occasional pigmented spots are observed in other

activity. However, one dose of the Mitfmi-sp _mutant

pro-tein is clearly not sufficient for proper melanocyte de- body regions, including the rump. Although the eyes

are smaller than normal, appear red, and lack pigment velopment since heteroallelic combinations involving

Mitfmi-vga9 _{and Mitf}mi-ew _{result in defective pigmentation altogether, they are not as severely affected as animals}

carrying the loss-of-function Mitfmi-vga9 _{mutation or in}

(Table 2, Figures 1 and 2). Interestingly, although the

homozygous phenotypes of the Mitfmi-vga9 _{and Mitf}mi-ew _MitfMi-wh_{animals (Table 2; Figure 3A). TheMitf}mi-rw

muta-tion is caused by the lack of a large pormuta-tion of theMitf

mutations are almost identical, the heteroallelic

combi-nations differ in that Mitfmi-vga9_/Mitfmi-sp _{animals have regulatory region, resulting in the absence of the 5}⬘

exons 1h and 1b and in aberrant expression of the gene

large pigmented spots while Mitfmi-ew_/

Mitfmi-sp _animals

have a uniformly gray coat color over most of their body; (Steingrı´mssonet al. 1994;Hallssonet al. 2000). Most

heteroallelic combinations involving Mitfmi-rw _{result in}

head and feet are white. This reflects the fact that the

Mitfmi-vga9 _{mutation is a loss-of-function mutation; the white microphthalmic animals: Combinations with the}

Mitfmi_,

MitfMi-or_{, and}

Mitfmi-ew_{mutations all produce white}

heteroallelic combination reveals the function of a

sin-gle copy of the Mitfmi-sp _{protein. The Mitf}mi-ew_/Mitfmi-sp _{microphthalmic animals (Table 2; Figure 1G) and}

Mitfmi-rw_/Mitfmi-vga9_{animals are white and microphthalmic}

combination, on the other hand, reveals the partial

dominant-negative nature of the Mitfmi-ew _{protein. Al- and most have a small pigmented head spot (Table 2;}

Figure 2). In all these cases the microphthalmia

ob-though theMitfmi-ew_{mutation behaves in a recessive}

fash-ion on its own and is classified as such in this study,in served is more severe than that inMitfmi-rw

homozygotes.

However, combinations of the Mitfmi-rw _{mutation with}

vitrostudies suggest that the mutant protein has strong

dominant-negative activity (Hemesath et al. 1994). MitfMi-b_andMitfMi-wh_{result in somewhat milder}

pheno-types: Mitfmi-rw_/MitfMi-b _{animals are white with an}

occa-However, the Mitfmi-ew _{protein is unable to enter the}

nucleus efficiently (Takebayashiet al. 1996;Nakayama sional pigmented spot and normal eye size (Table 2) while

Mitfmi-rw_/MitfMi-wh_{animals are white with a tan head spot;}

et al. 1998), resulting in a dominant-negative

cyto-plasmic protein. Since the protein needs to enter the eye size is near normal and lacks pigment (Figure 1H).

In all the cases described above, coat color is most nucleus to realize its effects, this may result in only

minor dominant-negative effectsin vivo, at least in some sensitive to mutations atMitf, indicating that

melano-cytes have the highest requirement for Mitf function.

Eye color follows, then eye size, and only a few of the notypes in homozygous condition, the third (Mitfmi-rw_{) is}

more severely affected; all three have milder phenotypes mutations show osteopetrosis. Thus, the pigment cells

of the retinal epithelium of the eye have a lower require- than that of MitfMi-wh _{in homozygous condition. Both}

semidominant and recessive mutations are

comple-ment forMitfprotein than do melanocytes, and

osteo-clasts seem to be able to function with very low amounts mented while only recessive mutations fail to be

comple-(if any) ofMitfin the cell. mented (Tables 1 and 2).

Interallelic complementation at Mitf: In contrast to This is further supported by previously reported

het-the results described above, most of het-the combinations eroallelic combinations involvingMitfMi-wh_{. For example,}

involving the MitfMi-wh _{allele show interallelic}

comple-Mitfmi-bw_/

MitfMi-wh_{are white with some pale yellow spots,}

mentation in which the resulting phenotype is more which depigment with age, whereas homozygotes for

normal than that of each of the homozygotes alone. This the recessiveMitfmi-bw_{mutation are white with black eyes}

is true for combinations involving the semidominant (Schaible 1963). The Mitfmi-ws _{mutation is}

semidomi-mutations Mitfmi_,

MitfMi-or_{, and}

MitfMi-b_{, as well as the}

nant; heterozygotes have a white belly spot while

homo-recessiveMitfmi-ew_{mutation and the}

Mitfmi-vga9

loss-of-func-zygotes are white, most with normal eye size.

Heteroal-tion mutaHeteroal-tion. All these combinaHeteroal-tions, exceptMitfMi-wh_/

lelic combinations of this mutation withMitfMi-wh_result

MitfMi-b_{, result in white animals with normal eye size}

in spotted animals with yellowish-brown-to-gray spots

(Table 2; Figure 3, A–E). The normal eye size is in sharp that depigment with age (Hollander1968). Thus, coat

contrast to the severe microphthalmia observed in each color is complemented. TheMitfmi-di_andMitfmi-ce

muta-of the MitfMi-wh_{, Mitf}mi_{, Mitf}Mi-or_{, Mitf}mi-ew_{, and Mitf}mi-vga9

tions have very similar phenotypes in homozygous

con-homozygotes (Table 2; Figures 2 and 3). The heteroal- dition (white coat and small eyes) and, in fact, the

mo-lelic combination ofMitfMi-wh_andMitfMi-b_{results in}

light-lecular defect involved is identical (Hallsson et al.

tan animals with occasional white spots (Figure 3F); _{2000). Consistent with this, heterozygous combinations}

while eye size is normal, the RPE layer contains some _ofMitfmi-di_andMitfmi-ce_withMitfMi-wh_{result in normal eye}

pigment (data not shown). Although the eye size of this _{size, showing that both mutations are complemented}

heteroallelic combination is more normal than that of _{(Westet al. 1985; M. L.Lamoreux, unpublished}

obser-MitfMi-wh_{homozygotes,Mitf}Mi-b_/MitfMi-b_{animals have}

nor-vations). mal eye size. Thus, the eye size of the heteroallelic

com-bination is similar to that of one of the homozygotes so

no complementation is observed with respect to this _DISCUSSION

feature. However, coat color pigmentation is more

nor-The allelic series at the Mitf locus shows that the

mal in the heteroallelic combination than in each of the

three major cell types affected by mutations at the locus, homozygotes, suggesting complementation with respect

melanocytes, osteoclasts, and retinal pigment epithelial

to this phenotype. Clearly, the MitfMi-wh _{mutation can}

cells of the eye, have different requirements for the Mitf complement both the microphthalmia and the coat

protein: Most of theMitfmutations affect pigmentation

color phenotypes of the differentMitfmutations.

of the coat, many affect eye development, and only a Interestingly, three recessive mutations are not

com-few result in osteopetrosis. Clearly, the requirement for

plemented byMitfMi-wh _{(Table 2). As explained above,}

Mitffunction is very different in the cell types in which

the heteroallelic combination MitfMi-wh_/Mitfmi-sp _results

the highest requirement is observed in melanocytes and in tan animals with occasional white spot. This

pheno-the lowest in osteoclasts. Osteopetrosis is observed only type is intermediate between the two homozygotes and

in homozygotes for theMitfmi_andMitfMi-or_{mutations as}

therefore no complementation is observed (Table 2).

well as in compound heterozygotes of these mutations

MitfMi-wh_/Mitfmi-rw_{animals also have an intermediate}

phe-(Table 2). No osteopetrosis is observed inMitfMi-wh

ho-notype although some complementation may be

ob-mozygotes or in any of the other mutations tested, ex-served with respect to eye development. In these animals

ceptMitfmi-ew_{, which shows mild hyperosteosis in the}

ho-coat color is white with tan head spots while eye size

mozygous condition (Steingrı´msson et al. 2002).

varies from microphthalmic to near normal (Figure

Although theMitfmi-ew_{mutation is classified as recessive}

1H); the difference in eye size can be bilateral with

in this study, the mutant protein has partial dominant-one eye near normal and the other microphthalmic.

negative activity, at least in osteoclasts. Recent studies

Perhaps most interestingly, the MitfMi-wh_/Mitfmi-bws

com-have shown that although osteoclasts are normal inMitf

pound heterozygotes have an intermediate phenotype

null mice as well as in mice carrying a loss-of-function showing tan spots and normal eye size (Figure 3H). In

mutation in the Mitf-related gene Tfe3, the combined

the homozygous condition theMitfmi-bws_{mutation results}

loss of both genes results in severe osteopetrosis (

Stein-in large, heavily pigmented spots (Table 1, Figure 3G)

grı´msson et al. 2002). Thus, the Mitf-associated os-and normal eye size. Thus, the phenotype of the

hetero-teopetrosis is seen only upon simultaneous loss of Mitf allelic combination is not complemented. While two of

and Tfe3 activity in osteoclasts. Only dominant-negative the recessive mutations that fail to be complemented

show this phenotype. The osteoclast phenotype never fail to be complemented byMitf are mutations that affect regulatory regions as well as mutations that affect shows interallelic complementation.

The phenotype ofMitfMi-wh_{mutant animals is unusual protein-coding regions. The common theme among the}

mutations that fail to be complemented is that they have in that heterozygotes are severely affected while

homozy-gotes have an intermediate phenotype compared to that milder effects in the homozygous condition thanMitfMi-wh

and the mutations that are complemented byMitfMi-wh_.

of the other Mitf alleles: In heterozygotes coat color

is severely affected while eye development is not; in This does not fit the criteria of transvection.

Despite the fact that the MITF protein can form both homozygotes, coat color is still severely affected while

eye development has an intermediate phenotype. This homo- and heterodimers, it is difficult to see how the

Mitf-associated complementation could be due to the

suggests different effects of the mutation in the two

pigment cells affected, melanocytes and RPE cells. Fur- formation of functional dimers by two different mutant

proteins. Three of the mutations that show interallelic thermore, it suggests that the dominant-negative nature

of theMitfMi-wh_{mutation has much more serious effects complementation with}

MitfMi-wh ₍

Mitfmi-ew_,

Mitfmi-mi_{, and} MitfMi-or_{) all affect the DNA-binding basic domain, the}

in melanocytes than in RPE cells.

In the eye, complementation is observed in many very same domain affected by theMitfMi-wh_mutation

(Ta-ble 1;Steingrı´mssonet al. 1994). The fourth mutation

different combinations withMitfMi-wh_{, even inMitf}Mi-wh_/

Mitfmi-vga9 _{animals in which the Mitf}Mi-wh _{allele is paired to show interallelic complementation isMitf}mi-b_{, whose}

molecular defect is in the loop of the HLH domain and with a loss-of-function mutation. Coat color is

comple-mented only whenMitfMi-wh_{is paired with hypomorphic also affects DNA-binding abilities of the protein (Table}

1;Steingrı´mssonet al. 1996). Thus, all these mutations

mutations such as MitfMi-b _{and Mitf}mi-ws_{, which already}

have normal eye development (Hollander1964). This affect DNA binding. Most interestingly, the

loss-of-func-tion mutaloss-of-func-tion Mitfmi-vga9 _{can complement the Mitf}Mi-wh

suggests that the complementation occurs in a reverse

allelic series, again reflecting the activity requirement mutation, suggesting that for complementation to take

place it is better to have no or very little Mitf activity

for Mitf in the different cell types. The threshold for

theMitfrequirement is lower in RPE cells than in mela- with theMitfMi-wh_{mutation than to have partial function.}

These facts are difficult to reconcile with the model of nocytes and therefore the eye phenotype is more easily

complemented than the coat color phenotype. Interest- complementation by active dimers.

Our observations lead us to propose an alternative ingly, no complementation is observed in combinations

with alleles with a phenotype more normal than that of dose-dependent model of interallelic complementation

(Figure 4). According to this model, the phenotype

MitfMi-wh_{in homozygous condition.}

To date, two main models have been proposed to is determined by the level of the MitfMi-wh _{protein in}

combination with a cell-type-specific level of sensitivity explain interallelic complementation. The first model

involves transvection in which one allele affects the ex- to this protein. Earlier studies have shown that like

wild-type Mitf, the MitfMi-wh _{protein comes in two distinct}

pression of a second allele on the homologous

chromo-some. Generally, these involve combinations of a regula- isoforms, which differ in the presence or absence of six

residues upstream of the basic domain (Steingrı´msson

tory mutation with a mutation in the coding region.

Transvection depends on chromosomal pairing and et al. 1994). The alternative six amino acids are the result

of alternative splice acceptor sites in exon 6; all tissues chromosomal rearrangements have been shown to

in-terfere with the complementation. The best examples analyzed so far contain both splice forms (Hallsson

et al. 2000). In vitro, the MitfMi-wh _{protein lacking the}

of this involve theUltrabithorax(Ubx) gene in Drosophila

where pairing of certain alleles results in partial comple- alternative six amino acids acts in a dominant-negative

fashion; the protein can dimerize with partner proteins

mentation of the phenotype (reviewed by Pirrotta

1999). The second model for interallelic complementa- but cannot bind DNA and interferes with the DNA

bind-ing of the related Tfe3 protein in vitro (Hemesath et

tion involves dimer formation between protein

mono-mers expressed by two different alleles. The different al. 1994). However, although the MitfMi-wh_protein

con-taining the alternative six amino acids cannot bind DNA alleles affect different functional domains and the

re-sulting dimer is therefore partially functional and com- as a homodimer, it can bind DNA as a heterodimer

with Tfe3 at levels similar to the wild-type Mitf protein plementation is observed. Examples of this include the

Egfrlocus in Drosophila (Razet al. 1991) and thelet-23 (Hemesathet al. 1994). Thus, the mutation produces

two different proteins with critically altered function: a

gene inCaenorhabditis elegans(Aroianet al. 1994).

In the case ofMitfreported here, neither of these two dominant-negative protein [MitfMi-wh₍⫺_{6)] and a}

pro-tein that cannot bind DNA as a homodimer, yet can models clearly apply. Transvection is a highly unlikely

explanation forMitf-associated interallelic complemen- bind DNA as a heterodimer [MitfMi-wh₍⫹_{6)] (Figure 4A)}

with its partners. Due to the intermediate phenotype of tation. The only allele that results in complementation

(MitfMi-wh_{) does not detectably affect regulation of the Mitf}Mi-wh _{homozygous animals, one of these proteins}

must be partly functional. gene and is a mutation in the protein-coding region

Figure4.—The biochemi-cal behavior of the MitfMi-wh protein provides a model for the interallelic complementa-tion at Mitf. (A) The bio-chemical behavior of the two different MitfMi-wh _proteins. (B) A model depicting the ef-fects of the mutant proteins on the phenotype. Yellow bars represent levels of nor-mal Mitf function while red bars indicate levels of neo-morphic activity. The dotted lines indicate threshold re-quirements for each type of activity. The thresholds can be different in the different cell types affected. Three dif-ferent situations are shown: wild type, MitfMi-wh

homo-zygotes, and the MitfMi-wh_/ Mitfmi-vga9_{heteroallelic}

combi-nation. In wild type (left), two different messages are expressed in all tissues analyzed, leading to the synthesis of two different Mitf proteins: The Mitf(⫺6) protein has 20% less stability in a complex with DNA than the Mitf(⫹6) protein (Hemesathet al. 1994). InMitfMi-wh_homozygotes

(middle), the MitfMi-wh(⫹6) protein is more or less normal as a heterodimer while the MitfMi-wh(⫺6) protein cannot bind DNA as a homo- or heterodimer. In addition, it has acquired new (neomorphic) properties, which negatively affect the phenotype. The resulting phenotype is a trade-off between the two different effects and differs in cell types. In the MitfMi-wh_/Mitfmi-vga9

heteroallelic combination (right) the dose of neomorphic activity has been reduced by one-half such that it is below the threshold required to see effects on the phenotype. At the same time, the normal activity of the MitfMi-wh(⫹6) protein is sufficient to allow normal function and complementation is observed.

protein is likely to provide the explanation for both From the mutant phenotype it is clear that the effects

of theMitfMi-wh_{mutation are relatively more serious in}

the interallelic complementation associated with this

mutation and the fact thatMitfMi-wh_{has the most severe the neural-crest-derived melanocytes than in RPE cells}

or osteoclasts. Coat color is severely affected inMitfMi-wh

phenotype in heterozygous condition. We therefore

propose that the genetic behavior of this mutation is homo- and heterozygotes while only homozygotes show

intermediate microphthalmia; bone development is due to the relative effects of homo- and heterodimers

involving this mutation where the MitfMi-wh₍⫺_{6) protein normal in both cases. This suggests a tissue-specific}

dif-ference in the effects of theMitfMi-wh_{mutation. Perhaps}

is very efficient as a dominant-negative protein and

where MitfMi-wh_{(⫹6) is at least partly functional. The two this is an indication that the mutant protein interacts}

with one or more melanocyte-specific proteins. Thus,

different versions of the MitfMi-wh _{protein may not be}

able to form DNA-binding dimers between themselves in MitfMi-wh_/⫹ _{animals, the strong dominant-negative}

action of the MitfMi-wh₍⫺_{6) protein may interfere with}

but may be able to dimerize with other partner proteins

in the cell. Furthermore, we propose that one of the two partner proteins in melanocytes. Alternatively, the new

(neomorphic) activity may negatively affect the function

isoforms of the MitfMi-wh_{protein (or both) has acquired a}

new (neomorphic) function, resulting in negative ef- of melanocyte-specific factors or processes. Together,

these effects result in the severe coat color phenotype fects in the cell. This new action may be the result of

dominant-negative action of the MitfMi-wh₍⫺_{6) protein observed in heterozygotes. Although the RPE cells of}

MitfMi-wh_/⫹ _and

MitfMi-wh_/

MitfMi-wh _{animals also contain}

against essential proteins or pathways in the cell. It may

also be due to a novel action of the MitfMi-wh₍⫹_{6) protein, the neomorphic activity (since they also express the}

mutantMitfgene), RPE cells are not as severely affected

e.g., binding the wrong promoter sequence or activating

the wrong set of genes with subsequent negative effects. since the activity threshold is different (Figure 4B) and

the RPE cells do not express the melanocyte-specific For the following discussion we assume that the new

(neomorphic) activity is associated with the MitfMi-wh _{protein(s) against which the dominant-negative Mitf}Mi-wh

(⫺6) protein acts.

(⫺6) protein. Finally, we propose that the different

activities of the two forms of the MitfMi-wh_{protein each The intermediate eye phenotype observed in Mitf}Mi-wh

homozygotes supports the idea that the MitfMi-wh₍⫹₆₎

have their own threshold requirements in the different

cell types affected. A model depicting theMitf-associated protein has partially normal function in RPE cells. The

partially normal function of the MitfMi-wh_{(⫹6) protein}

is unaffected by the dominant-negative activity of the

at themilocus. J. Biol. Chem.268:20687–20690. MitfMi-wh₍⫺_{6) protein since Mitf}Mi-wh₍⫹_{6) is unable to form}

Konyukhov, B. V., andV. V. Osipov, 1968 Interallelic

complemen-tation of microphthalmia and white genes in mice. Genetika4:

DNA-binding homodimers with other mutant MitfMi-wh

65–76.

proteins (Hemesathet al.1994). In the different

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allelic combinations, the new (neomorphic) action of _{1992 Thevitgene maps to themi(Micropthalmia) locus of the}

laboratory mouse. J. Hered.83:435–439.

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Lerner, A. B., T. Shiohara, R. E. Boissy, K. A. Jacobson, M. L. of MitfMi-wh _{homozygotes, thereby reducing the effects}

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Genet.11:442–448. (Figure 4B). Thus, the heteroallelic combination of

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88:2488–2494. to generate eyes of normal size. In heteroallelic

combi-Motyckova, G., K. N. Weilbaecher, M. Horstmann, D. J. Rieman,

nations with milderMitfmutations that still retain some _{D. Z. Fisheret al., 2001 Linking osteopetrosis and}

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microphthal-Mitf activity, the dominant-negative MitfMi-wh₍⫺₆₎

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tein interferes with the function of the mutant proteins

5798–5803.

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Hodgkinsonet al., 1998 Mutations inmicrophthalmia, the mouse

morphic activity is still effective, resulting in no

comple-homolog of the human deafness geneMITF, affect neuroepithe-mentation.

lial and neural crest-derived melanocytes differently. Mech. Dev.

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Pirrotta, V., 1999 Transvection and chromosomal

trans-interac-differently by the two versions of the MitfMi-wh _protein

tion effects. Biochim. Biophys. Acta1424:M1–M8. than by the corresponding versions of wild-type Mitf

Raz, E., E. SchejterandB. Z. Shilo, 1991 Interallelic

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pro-191–201. teins on well-defined promoter elements in the different

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iden-by the National Cancer Institute (N.G.C. and N.A.J.), the National

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