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microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.

microphthalmia, a critical factor in melanocyte development, defines a discrete transcription... Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family Timothy J. Hemesath / Eirikur Steingrimsson,^ Gael McGill / Michael J. Hansen / James Vaught/ Colin A. Hodgkinson,^ Heinz Amheiter,^ Neal G. Copeland/ Nancy A. Jenkins/ and David E. Fisher^'* ^Division of Pediatric Hematology/Oncology, Dana Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 USA; ^Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 USA; ^Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 USA The ndcrophtbalinia (mi) gene appeals essential foi pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities. [Key Words: microphthalmia; dimerization; DNA binding; M box; b-HLH-ZIP] Received August 23, 1994; accepted in revised form October 11, 1994. of melanocyte growth or survival. The mi gene was A striking inheritable disorder of development in the cloned recently and shown to predict a basic/helix- mouse is microphthalmia [mi], a syndrome first recog­ loop-helix/leucine zipper (b-HLH-ZIP) factor (Hodgkin- nized >50 years ago as a coat color mutation (Hertwig son et al. 1993; Hughes et al. 1993; Tachibana et al. 1942). The human mi gene has also been linked compel- 1994). Mammahan b-HLH-ZIP factors and the related lingly to the human pigment cell disorder Waardenburg b-HLH group contain several important regulators of Syndrome (Hughes et al. 1994). Mutations at the mouse cell proliferation and development such as Myc/Max mi locus result in pigment cell defects in the skin (pro­ (Blackwood and Eisenman 1991; Prendergast et al. 1991) ducing white spotting), eyes (producing small eyes), and and MyoD-related factors (for review, see Olson 1990, inner ears (resulting in deafness). Mast cell defects have and references therein; Weintraub 1994). Given the ef­ also been recognized for certain mi alleles, a pattern re­ fects of mi mutations on melanocyte biology, mi may sembling the melanocyte/mast cell pattern of SI/kit-de­ regulate comparable pathways in melanocytes. Although fective mice and suggesting a coimection between these a significant number of b-HLH-ZIP factors are known, factors in signaling (Dubreuil et al. 1991; Ebi et al. 1992). biological activities are clear for only a few, primarily Bone resorption and other neural crest or neuroepithelial defects have also been observed for certain mi alleles (for Myc and its related partners (see Prendergast and Ziff review, see Green 1989), suggesting that Mi protein may 1992; Ayer and Eiseimian 1993; Zervos 1993). Only very function in part through oligomeric interactions with few candidate target genes have been identified so far other factors. that are regulated by these factors. The striking biologi­ cal consequences of mi mutations suggest a major role in The devastating consequences of mi mutations on me­ melanocyte development and even point to specific can­ lanocyte development suggest that Mi is a key regulator didate target genes. Melanocytes represent a neural crest-derived lineage *Conesponding author. whose pigmentation function is easily assessed because GENES & DEVELOPMENT 8:2770-2780 © 1994 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/94 $5.00 2770 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein and protein-DNA interactions have been revealed melanocytes are not critical to survival of the whole an­ through a characterization of the proteins encoded by imal. A great deal has been learned about pigmentation seven mutant mi mouse alleles (Steingrimsson et al. enzymes and their regulation through study, for exam­ 1994). These mutations cluster within or near the ple, of albino (tyrosinase) mutants (for review, see Hala- b-HLH-ZIP motif of mi and display striking effects on ban and Moellmann 1993). Determination of regulatory heterodimerization and DNA binding that largely ex­ elements critical for pigmentation have revealed an 11- plain the unique severity and inheritance patterns of the bp sequence known as the M box containing the core different mi mouse strains. Novel structural features of element CATGTG, which is highly conserved in the pro­ b-HLH-ZIP biochemistry have also been revealed, such moters of the three major pigmentation enzyme genes as the surprising ability of an alternative exon outside of tyrosinase, and tyrosinase-related proteins 1 and 2 the basic domain to modulate b-HLH-ZIP-dependent (Shibahara et al. 1991; Lowings et al. 1992; Yavuzer and DNA recognition. Finally, Mi was shown to transcrip­ Coding 1994). The presence of tissue-specific transcrip­ tionally activate a reporter driven by the pigmentation tion factors capable of interacting with and transcrip­ promoter M-box element, suggesting that this family of tionally activating elements such as this may shed light factors plays a central role in the tissue-specific devel­ on the regulation of pigmentation and perhaps other me- opment of melanocytes. lanocyte-specific functions. The opportunity to examine structure/function rela­ tionships for transcription factor mutations coordinately in vitro and in mice has rarely been possible. The mul­ Results titude of mi mouse mutants provides a unique opportu­ DNA recognition by Mi nity to examine biochemical consequences of biologi­ cally important b-HLH-ZIP mutations. The b-HLH- The Mi protein produces a gel shift complex (Fig. 1) with ZIP family contains a short ~20-amino-acid basic DNA containing the CACGTG hexanucleotide derived domain rich in basic amino acids that makes sequence- from adenovirus major late promoter (MLP). Several de­ specific DNA contacts. Carboxy-terminal to it is the letions were made to determine the protein domains re­ HLH-ZIP containing two amphipathic helices separated quired for DNA binding (Fig. 1 A). It was possible to trun­ by a flexible loop and a carboxy-terminal leucine zipper. cate from the amino terminus to the beginning of the The HLH-ZIP mediates dimeric interactions necessary basic domain and from the carboxyl terminus to the end for DNA binding (Ferre-D'Amare et al. 1993). Restricted of the leucine zipper domain without loss of DNA bind­ heterodimerization plays a major role in regulating de­ ing (Fig. IB, lanes 2-4). Further deletion from the car­ velopmental programs ranging from inhibition of myo- boxyl terminus removed part of the leucine zipper and genesis by the HLH protein Id (Benezra et al. 1990) or abolished DNA binding (Fig. IB, lane 5). Therefore, the morphogenesis by extiamachiocaete (Ellis et al. 1990; leucine zipper was essential for stable complex forma­ Garrel and Modolell 1990) to cooperation in cellular tion. transformation by Myc/Max (Blackwood and Eisenman Sequence specificity of DNA recognition was verified 1991; Prendergast et al. 1991; Kato et al. 1992; Amati et by competition analysis using both CACGTG (Fig. IC) al. 1993). The ability to group these factors into families, and CATGTG (Fig. ID) probes. In each case, a double based on dimerization specificities, provides a useful point mutant (GAGGTG) failed to compete the specific handle for analysis of their biological roles. complex at concentrations effectively competed by un­ Most b-HLH-ZIP proteins recognize the hexamer core labeled CACGTG competitor. sequence CACGTG or the related sequence CATGTG, whereas AP-4 (Hu et al. 1990) and most b-HLH proteins recognize CAGNTG hexamers. A hexamer containing Mi is a member of a discrete family of b-HLH-ZIP the CATGTG sequence is present in the mouse immu­ factors noglobulin heavy chain enhancer and was used to isolate and characterize the transcription factor TFE3 (Beck- Stoichiometry of protein to DNA in the bound com­ maim et al. 1990; Roman et al. 1992). Although most plexes was examined by mixing full-length Mi protein b-HLH-ZIP factors interact avidly with cognate targets, with the isolated b-HLH-ZIP region (Fig. 2, lanes 2 and it has been difficult to elucidate tissue-specific activities, 3, respectively). A single new intermediate mobility gel in part because most of these factors are expressed ubiq­ shift complex was observed (Fig. 2, lane 4). Overexposure uitously. In this regard, mi, which is tissue restricted, is failed to reveal additional intermediate complexes, sug­ an attractive candidate as an M-box activator and regu­ gesting that the protein-DNA stoichiometry is 2:1. lator of pigmentation gene expression. Experiments were also imdertaken to determine The studies described here identify Mi's DNA-binding whether Mi is capable of forming DNA-binding het- activity and its ability to form stable DNA-binding het- erodimers with several other b-HLH-ZIP proteins. Only erodimers with TFEB, TFE3, and TFEC, three other three proteins, TFEB, TFE3, and TFEC, were found to b-HLH-ZIP factors. Collectively, these four proteins form intermediate mobility complexes with Mi (Fig. 2, comprise a distinct family that likely modulates the bi­ lanes 5-13). In these mixing experiments TFE3 (but not ological activity of Mi through hetero-oligomer forma­ Mi) preferentially heterodimerizes, probably reflecting tion. The biological importance of Mi's protein-protein different kinetics from Mi. In contrast, no heterodimers GENES & DEVELOPMENT 2771 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. A alt I .. V exo . n bHLHZIP 419 aa NA192 OO CD CA261 CA308 S < O Competitor: GAGGTG CACGTG CA319 < ^, Ol CJ CL o o 2 = ?5 °2 N (ng) o o o o ■> — T — CC 3 <i <] o5 i n CM i n (N D Competitor: CACGTG GAGGTG ■o UL 6 (ng) 1 10 30 50 1 10 30 50 » 1- CATGTG Free probe CACGTG DNA probe 1 2 3 4 5 6 7 8 1 2 3 4 5 4 5 6 1 2 3 Figure 1. mi gene and deletion analysis. (A) Schematic representation of the Mi protein. Hatched areas depict basic helix-loop-helix, and leucine zipper domains (b-HLH-ZIP). Positions of the various amino-terminal and carboxy-terminal deletions are indicated using numbering based on the melanocyte form of Mi that contains 419 amino acids (Hodgkinson et al. 1993; Steingrimsson et al. 1994). {B) DNA binding by Mi. Radiolabeled DNA containing the core hexanucleotide CACGTG was used in gel shift analysis. Proteins were derived from in vitro translation using reticulocyte lysate and were either full length or truncated as indicated. Endogenous reticu­ locyte DNA binding is indicated by an asterisk (*).(- RNA) Reticulocyte lysate unprogrammed by exogenous RNA. (C) Competition for CACGTG probe. Mi protein containing an intact b-HLH-ZIP region (truncation NA192;CA308) was tested for DNA binding to radiolabeled CACGTG probe. Unlabeled competitor DNA fragments of identical size were added as indicated in units of nanograms. Core sequences of the competitors are shown. (*) A background reticulocyte activity; {—>■] Mi specific activity. [D] Competition for CATGTG probe. Purified recombinant Mi protein was bound to radiolabeled |J.E3 probe DNA in the presence of unlabeled competitor DNAs as indicated, (^►j The Mi specific activity; (*) a contaminating activity present in the probe. were observed upon mixing Mi with E47S (Fig. 2, lanes produced and tested directly for their ability to bind 14-16), Max, Myc, upstream stimulatory factor (USF), or DN A as homodimers or as heterodimers with TFE3. several non-HLH-containing transcription factors (data Identical result s wer e obtained whe n heterodimerization no t shown). Therefore, of the known and tested candi­ was tested with wild-type Mi, TFEB, or TFEC (data not date partners. Mi appears to be capable of forming stable shown). The seven mutations and their properties are DNA-bindin g heterodimers wit h only TFEB, TFEC, and summarize d in Table 1. TFE3. With the additional observation that TFEB and Whe n tested for homodimeric DN A binding, all three TFEC form stable heterodimer s (Fig. 2), all combinations semidominan t and two recessive mutan t proteins failed of these four proteins have now been shown to het- to bind DN A (Fig. 3A, lanes 3-8). Only the helix 1 mu­ erodimerize with one another but not with any other tan t D222N (mi^") mutan t protein, which is inherited know n b-HLH-ZIP proteins (Fig. 2; Fisher et al. 1991; recessively, appeared to bind DN A normally. Quantita­ Zha o et al. 1993), indicating that they constitute a dis­ tive affinity measurements revealed mi'' " t o bind wit h a crete group of interactive proteins, which we refer to as K^ only 6% greater than that of wild-type Mi (using th e MiT family. forms containing the 6-residue alternative insert), a dif­ ference within the 10% standard error of our measure­ ment s (data not shown). When mixed with TFE3, mi'*'" Mutant alleles affect MiT interactions wa s the only mutant protein able to produce a het- Recen t molecular genetic studies of Steingrimsson et al. erodimeric complex wit h TFE3 (Fig. 3A, lanes 10-16). (1994) suggest tha t dominant-negative Mi mutation s are Examinatio n of heterodimer mixing experiments us­ dominantl y inherited whil e regulatory mutation s or mu­ ing th e mutan t Mi proteins (Fig. 3A) revealed a striking tation s that prevent or reduce Mi protein dimerization loss of TFE3 homodime r activity in man y reactions. Ad­ are recessively inherited. To examine this possibility, dition of mi. Mi""", Mi"^^, or mi^^ proteins essentially mutan t proteins corresponding to the seven mi muta­ ablated TFE3 homodimeric DNA-binding activity (Fig. tion s characterized by Steingrimsson et al. (1994) were 3A, lanes 12,13,15,16). In contrast, the recessive allele GENES &, DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein < Mi/Mi TFEB/Mi TFE3/Mi Mi/TFEC E47/Mi TFEC/TFEB protein-protei n interactions, coimmunoprecipitations were performed using ^^S-labeled mutant Mi proteins, '^IM B TMB TMB MT B EiB TcTbB unlabeled recombinant TFEB, and a TFEB-specific anti­ mt'-mM *"»"""'wi iP*'W ..|«||| ^^^^ body. Antibod y specificity was verified by supershift of a TFEB/DN A complex but failure to supershift Mi or ■ " ■ • ■■ ■ ^" fMj othe r b-HLH-ZI P proteins (data not shown). Specificity wa s indicated further by the dependence for TFEB i n the Mi Mi HI Mi Mi V' I coimmunoprecipitation s (Fig. 3B, lanes 1,2) as well as th e dependence of antibody (data not shown). The la­ beled Mi protein migrates as a doublet of —15 Kd. TFEB- specific antibodies coimmunoprecipitated wild-type Mi, th e three semidominant proteins mi, Mi°'', and Mi"^"^, as wel l as the recessive protein mi'*''* (Fig. 3B, lanes 2-6), consisten t with a dominant-negative inhibition of DN A 1 2 3 4 5 6 7 8 9 10 11 12 13 141516 17 18 19 binding by the products of the semidominant alleles. A Figure 2. Mi forms stable heterodimers with TFEB, TFE3, and similar coimmunoprecipitation pattern was also ob­ TFEC. Full-length and truncated forms of various b-HLH- served for mi^'^ (data no t shown). Th e zipperless reces­ ZIP proteins were translated separately in vitro and equivalent sive protein mi''^ did not efficiently coprecipitate (Fig. volumes were mixed (post-translationally) prior to the addi­ 3B, lane 7), althoug h a weak signal was observed, possi­ tion of radiolabeled CACGTG probe. (M) The Mi truncation bly reflecting a propensity to form HLH-mediated tet- NA192;CA308; (Mi) full-length Mi protein; (B) both proteins ramer s in the absence of DN A (Fisher et al. 1991; An- mixed together; (T) TFEB, TFE3, or TFEC as indicated; (Tc) thony-Cahil l et al. 1992; Farmer et al. 1992; Fairman et TFEC; (Tb) TFEB. The Mi used in lanes 3-10 encompasses the b-HLH-ZIP region; the Mi used in lanes 2 and 11-16 is full al. 1993). length. The fragment of TFEB used contains all but the first 265 amino acids (TFEB-AA265; Fisher et al. 1991). TFE3 is full- length (Beckmaim et al. 1990). The TFEC fragment contains the Alternative splice affects basic domain function isolated b-HLH-ZIP region (NA99;CA204). E47S is a truncation Th e mi message exists in splice forms either encoding or of E47 that includes the b-HLH region (Miirre et al. 1989). lacking 6 amino acids just amino-terminal to the basic domai n (Hodgkinson et al. 1993). Th e mf^ mutation af­ fects the polypyrimidine tract of the splice acceptor and mi''^ contains a stop codon tha t removes the leucine zip­ precludes formation of Mi protein containing the per and did no t affect TFE3-binding activity (Fig. 3A, lane 6-amino-acid insert (Steingrimsson et al. 1994). These 11). All three semidominant alleles contain basic do­ mic e produce normal pigment but exhibit a measurable mai n mutations, failed to bind as homodimers, and ad­ decrease in the pigmentation enzyme tyrosinase within ditionally suppressed DN A binding by TFE3 in an appar­ ski n (Wolfe and Coleman 1964). Despite the subtlety of entl y dominant-negative fashion. it s homozygous phenotype, the mi^^ allele enhances the effective phenotype of semidominant mi alleles in a Surprisingly, of the three recessive mutant proteins, compoun d heterozygote (Wolfe and Coleman 1964). To mi'' " bound DN A indistinguishably from wild-type pro­ examin e biochemical relevance of thi s alternative splice, tei n despite its helix 1 mutatio n (and the striking phe- wild-type Mi proteins with and without the 6-amino- notyp e of mi^^ mice) (Fig. 3A, lanes 6,14), suggesting acid insert were examined (Fig. 4A, lanes 2,3). Although tha t this mutation might disrupt a function other than th e two proteins boimd DNA similarly, quantitative DN A binding. Th e recessive allele, mi''^, contains a stop measurement s revealed that the splice form containing codon at the begiiming of the leucine zipper, failed to th e insert bound wit h 20% higher affinity than the form bind DNA, and was also incapable of suppressing the lacking th e insert (K^ = 290 and 349 JJLM, respectively , in DNA-bindin g activity of TFE3 (Fig. 3A, lanes 3,11) be­ presence of poly [d(I-C)]. N o large effect was observed for havin g "recessively" in vitro. A third recessive muta­ th e alternative 6-amino-acid insert on heterodimeric tion, mi^^, contains a 25-amino-acid deletion that re­ binding of wild-type Mi wit h TFE3 (Fig. 4A, lanes 4—6). move s the amino-terminal half of the basic region but Surprisingly, however, the presence of the 6-amino- does not involve the HLH-ZIP. This mutant failed to acid insert had a profound effect on DN A binding of the bind DNA as a homodimer and also suppressed the basic domain mutan t I212N (Mi^"^), th e allele that dis­ DNA-bindin g activity of TFE3 (Fig. 3A, lanes 8,16). As a basic domain deletion, this in vitro behavior was ex­ plays interallelic complementation. As shown in Figure pected for mi^^. Its recessive inheritance is surprising, 4B, presence of the insert restored heterodimeric DNA however. Importantly, this discrepancy between th e bio­ binding by Mi^'^ wit h a wild-type partner (Fig. 4B, lanes chemica l behavior of the mi^"^ protein and the genetic 1-4,9,11). In contrast, presence of the upstream insert behavior of th e mi^"^ allele suggests tha t these deleted 25 did not restore heterodimeric DN A binding for a differ­ en t basic region mutan t (mi), indicatin g th e specificity of amin o acids carry out a second function (aside from thi s effect for Mi"^^ (Fig. 4B, lanes 5-7,10). Thus, pres­ DN A binding). ence of th e upstrea m insert restored DN A binding to the To verify that the TFE3 suppression seen by proteins jyjjwh protein if the heterodimer partner was wild type. encoded by the semidominant alleles occurred through GENES & DEVELOPMENT 2773 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. Table 1. Miciophthalmia mutant alleles DNA binding Symbol Mutation^ Description homo- hetero-^ dom-neg*^ Semidominant microphthalmia mi no no yes del R217'* deletion within basic domain oak ridge Mi°' R216K no no yes basic domain mutation (facing DNA) white Mr^ I212N basic domain mutation (away from DNA) no yes^ yes* Recessive cloudy eyed no no no mf^ R263 STOP deletes leucine zipper and carboxyl terminus eyeless white mf"^ del A187-I212 deletion into basic domain no no yes vitiligo mr' D222N helix 1 mutation yes yes no Enhancing spotted mfP del 186-191 loss of alternative 6-amino-acid exon yes yes no Interallelic complementation white Mr'' I212N basic domain mutation (away from DNA) no yes* yes* ^Steingrimsson et al. (1994). ''Heterodimeric DNA binding tested with wild-type binding partners (TFE3 and Mi). '^Dominant-negative effects tested through inhibition of homodimeric wild-type protein in same reaction. ■^The mi allele deletes an Arg codon among a cluster of four in the basic domain. It is unclear which one has been deleted. e^^jwh mutant protein can bind as heterodimer with wild type only in presence of the 6-amino-acid upstream insert and suppresses (dom-neg) only in absence of insert. fjVlj«''i mutant protein is dominant negative only in the absence of the 6-amino-acid upstream insert. suggesting that this 6-amino-acid insert acts to stabihze family of proteins and whose complexity of allelic in­ the basic domain/DNA complex, hiterestingly, the teractions may be largely explained by these features. I212N mutation in the Mi"^*" protein is the only basic Biochemical analysis of Mi demonstrated its capacity to region mutant predicted to face away from DNA in the specifically recognize the DNA core sequences basic domain a-helix, on the solvent-exposed face (Ferre- CACCTC and CATGTG (Fig. 1). This DNA binding ap­ D'Amare et al. 1993; Fisher et al. 1993; Steingrimsson et peared to be dimeric based on mixing experiments that al. 1994). The restoration of DNA binding for Mi"^*" may result in the formation of a single intermediate mobility account for the interallelic complementation character­ complex (Fig. 2). Although this observation does not for­ istic of this allele. mally prove 2:1 stoichiometry of protein to DNA, the DNA cocrystallographic analyses of Max and USF showed dimeric protein interaction with the cognate Mi over expression tianscriptionally activates DNA template (Ferre-D'Amare et al. 1993, 1994). Addi­ an M box-driven reporter in fibroblasts tionally, the importance of Mi's leucine zipper was dem­ onstrated by the loss of DNA binding upon its deletion. We have tested the ability of mi to activate transcription A substantial body of data indicate that the leucine zip­ of a reporter driven by the M-box pigmentation gene pro­ per is necessary for dimerization and DNA binding by moter element (Shibahara et al. 1991; Lowings et al. b-HLH-ZIP proteins (Dang et al. 1989; Gregor et al. 1992; Yavuzer and Coding 1994) because of our demon­ 1990; Beckmaim and Kadesch 1991; Blackwood and stration that Mi is capable of binding its CATGTG core Eisenman 1991; Fisher et al. 1991; Prendergast et al. sequence in vitro (Fig. ID). Cotransfection of mi and the 1991; Blanar and Rutter 1992; Roman et al. 1992). M-box reporter into NIH-3T3 cells resulted in Mi-depen­ dent activation of the luciferase gene to levels > 13-fold above controls (Fig. 5). Stimulation of the luciferase ac­ Mi belongs to a discrete MiT family tivity was dependent on both the presence of the M-box element in the reporter construct and on the cotransfec­ Based on the phenotypic complexity of heterozygous tion of mi. Although identical to the immunoglobulin combinations of mi alleles (for review, see Green 1989), enhancer element |xE3 element at its core (CATGTG), it is likely that mi function depends on heterodimer for­ the M box differs in flanking positions, which are con­ mation during development. Heterodimeric DNA bind­ served from mouse to human in the three pigmentation ing was seen for Mi protein in combination with TFEB, TFE3, or TFEC (Fig. 2). With the observation that TFEB enzyme genes tyrosinase, and tyrosinase-related proteins and TFEC were also capable of heterodimerization and 1 and 2. Recognition of M-box elements by Mi may con­ DNA binding, all dimeric combinations of these factors stitute a critical component in the elaboration of mela- have now been demonstrated (Fig. 2; Fisher et al. 1991; nocyte-specific gene expression. Zhao et al. 1993). Heteromeric DNA-binding interac­ tions are otherwise quite restricted for these proteins, as Discussion none of them have been shown to heterodimerize with other HLH or HLH-ZIP factors. Whereas TFEB and TFE3 The experiments presented here demonstrate that the are ubiquitous factors (Beckmann et al. 1990; Carr and Mi protein is a transcription factor that forms homo- and Sharp 1990) and TFEC is tissue restricted (Zhao et al. heterodimeric DNA-binding complexes within a small 2774 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmi a protein importan t in the human pigmentation disorder Waar- denburg Syndrome^ which is dominantly inherited and ^ > LU I— was recently linked to th e huma n mi locus (Hughes et al. 1994). Th e behavior of these mutants , particularly those wit h unanticipated protein function, may aid in the ^migmm^S TFE3+ + LU h- I- hr h- I- U_ D C ':''&f. '_ wK~W^ 1 2345678 9 1011 1213U151 6 1 l-tb. - + + + + + + 1 2 3 4 5 6 TFE3 + TFE3 + ro + + 9HMMBI^$^^'^^ ^ : a: 14.3 ■fi t \^ ■§ s S ■f 5 £ 1 2 3 4 5 6 7 ¥" 3 Figure 3. DNA-binding properties of mi mutants. (4) Wild- type Mi (Mi-WT) and six different mutant Mi proteins were «?«» synthesized in vitro as amino-terminal deletions beginning with amino acid 109 (to visualize intermediate mobility com­ plexes). Proteins were tested in DNA-binding assays either alone (lanes 2-8] or in post-translational mixes with TFE3 (lanes 10-16]. The semidominant alleles mi, Mi°'', Mi^^, and the re­ cessive alleles mi"'', mi"\ and mi^^ were tested. The positions of two background reticulocyte bands are indicated (*), the lower one being remote from the strong signals and demonstrat­ ing evenness of sample loading, [B] Immunoprecipitation of wild-type (WT) and mutant Mi proteins with unlabeled recom­ 9 10 n 12 1 2 3 5 6 7 binant TFEB, using a TFEB specific antibody. Specificity is seen in lane 1, where lack of TFEB results in no coprecipitation. Figure 4. Alternative splice restores heteromeric DNA binding Wild-type Mi, the three dominant-negative proteins (mi, Mi°', by Mi'"'*. [A] DNA binding by two splice forms of Mi. Wild-type and Mi^*^), and mi"'^ coprecipitate efficiently with TFEB (lanes Mi protein (AA109;CA308) either lacking (WT - ) or containing 2-6); mi*^^, a zipperless protein, is very weakly coprecipitated, (WT +) the 6-amino-acid alternative exon was tested for DNA perhaps through a propensity to form HLH-dependent tetramers binding using the CACGTG probe, either alone or in the pres­ (lane 7). ence of TFE3. No obvious differences in DNA binding or het- erodimerization were apparent. Several background bands (*) represent reticulocyte proteins capable of DNA binding. [B] Six- 1993), i t wil l be importan t to determin e th e developmen­ amino-acid insert restores heterodimeric DNA binding by Mi^^. The basic domain mutant Mi*"" (AA109;CA308) was syn­ tal expression of these factors within cell lineages af­ thesized either with (Wh -I- j or without (Wh - ) the 6-amino-acid fected by mi mutations. Thus, these four proteins repre­ insert and tested for DNA binding (CACGTG) in the presence of sen t a distinc t Mi T family that likely participates in piv­ TFE3 (lanes 1-8] or alone (lane 9). A truncated form of Mi^'' otal developmental pathways, although other family contains only the b-HLH-ZIP (Wh-b). Another basic domain member s might exist as well. mutant, mi, was also synthesized from amino acid 109 (AA109;CA308) in the presence {mi + ] or absence (see Fig. 3) of the 6-amino-acid insert and tested for DNA binding with TFE3 Biochemical lesions and biological consequences (lane 6) or alone (lane 10]. Presence of the 6 amino acids restored We show here that dominant-negative protein behavior heterodimeric DNA binding to the Mi"^^ mutant (—>) without affecting the mi protein. Several background reticulocyte lysate appears to explain semidominant inheritance of mi alle­ bands are observed (see lane 12, unprogrammed lysate). les. This is relevant for mouse mi and is likely to be GENE S & DEVELOPMEN T 2775 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. Mi proteins producing recessive inheritance are also instructive regarding b-HLH-ZIP function and highlight functionally relevant regions unlikely to produce the S 150 dominant inheritance of Waardenburg Syndrome. Impor­ tantly, two (mi'^^ and mi^^] of the three display bio­ chemical behavior that is not expected. The third, mi'^^, introduces a stop codon at the carboxyl terminus of the HLH domain (Steingrimsson et al. 1994), thereby trun­ cating the leucine zipper. The transcription factor USF, however, appears to be capable of binding DNA without % 50 its leucine zipper (Gregor et al. 1990; Ferre-D'Amare et al. 1994). By failing to dimerize, the mi''^ protein should exert no dominant-negative effect at the level of DNA binding, as was observed in mixing experiments (Fig. 3). Luc + M-Luc + Luc + M-Luc + The weak coimmunoprecipitation of mi*^® by TFEB (Fig. vector vector Mi Mi 4) suggests that the HLH domain alone can measurably Figure 5. Mi stimulates transcription from a promoter con­ oligomerize, perhaps as a tetramer, in the absence of struct containing M-box elements. NIH-3T3 cells were tran­ DNA (Anthony-Cahill et al. 1992; Farmer et al. 1992; siently transfected and assayed after 24 hr for luciferase activity Fairman et al. 1993; Fisher et al. 1993). (expressed in relative light units). Error bars represent the stan­ dard deviation of triplicate samples. Transfected DNA con­ The mf^ allele predicts a 25-amino-acid deletion tained a luciferase reporter plasmid containing a minimal SV40 (Steingrimsson et al. 1994) that begins amino-terminal to promoter alone (Luc) or carrying four upstream copies of an (and deletes much of) the basic domain. This protein M-box element (M-Luc), and a CMV-driven expression vector failed to bind DNA as either a homodimer or heterodi- alone (vector) or containing a cDNA encoding wild-type Mi (Mi) mer. Like the semidominant alleles, it repressed DNA lacking the 6-amino-acid alternative insert. Weak M box-spe­ binding by wild-type protein because the HLH-ZIP do­ cific basal activity is seen in 3T3 cells as well as strong Mi- mains were intact. Interestingly, the mi^^ allele is in­ specific trans-activation. herited recessively suggesting that dominant-negative function is not fully realized in vivo. Potential explana­ identification and characterization of human mi lesions tions include the loss of a nuclear localization signal or capable of producing Waardenburg SyndromC; eventually decrease in protein stability. allowing for genetic screening in affected families. DNA The D222N mutations [mi^^] produces a helix 1 mu­ recognition by the basic domain can be disrupted in sev­ tation (Steingrimsson et al. 1994) with virtually no mea­ eral ways, some of which are reminiscent of the MyoD surable effect on DNA binding (Fig. 3) but produces pro­ inhibitor Id (Benezra et al. 1990) and the Diosophila fac­ gressive, aging-dependent melanocyte death (Lemer tor extiamacwchaete (Ellis et al. 1990; Garrel and Mo- 1986; Lemer et al. 1986). It is possible that the small dolell 1990). (6%) difference in K^ produced by this mutation is suf­ ficient to produce the aging-dependent vitiligo in these The basic domain of b-HLH-ZIP proteins recognizes mice. Altematively, this helix 1 mutation may affect DNA through a discrete a-helical face (Fisher et al. 1991) tetramerization, a property of many HLH proteins. TFEB that forms an iminterrupted structure with helix 1 of the has been shown previously to exist in a tetrameric state HLH domain (Ferre-D'Amare et al. 1993). This is an in­ in solution that dissociates into DNA-binding dimers trinsically unstable a-helix requiring DNA binding to upon addition of DNA (Fisher et al. 1991). Similar tet- stabilize its folding (Fisher et al. 1993; Ferre-D'Amare et ramers have been observed for several other HLH-con- al. 1994). The mi protein lacks a basic region arginine taining proteins including Myc (Dang et al. 1989), MyoD (Hodgkinson et al. 1993) which should shift the rota­ (Anthony-Cahill et al. 1992), and myogenin (Farmer et al. tional register of the basic domain a helix by —100° rel­ 1992). The Id protein's inhibition of MyoD DNA binding ative to the HLH, precluding DNA binding. The R215K appears to be mediated by tetrameric complexes (Fair- mutation in Mi°'' (Steingrimsson et al. 1994) destroys man et al. 1993) consistent with the observation that DNA binding in TFEB (Fisher et al. 1993) as well as in Mi tetrameric forms carmot bind DNA (Fisher et al. 1991). (Fig. 3). Although this position appeared to only make a The aspartate 222 mutated in mi"^* (Steingrimsson et al. phosphate contact in the cocrystal structure of Max/ 1994) is located within the four-helix bundle predicted DNA (Ferre-D'Amare et al. 1993), the fact that lysine could not substitute suggests another critical fimction, from the Max/DNA cocrystal structure (Ferre-D'Amare most likely including salt bridge formation with the up­ et al. 1993) and could participate in interhelical salt stream glutamate, thereby stabilizing a-helical folding. bridges, although its disruption does not appreciably af­ Surprisingly the semidominant mutation I212N {Mi^^] fect dimerization. is predicted to face away from the major groove of the DNA on the basic domain a-helix (Fisher et al. 1991, Alternative splice modulates DNA binding 1993; Ferre-D'Amare et al. 1993) and provides evidence that the basic domain is subject to significant regulatory Although b-HLH-ZIP DNA binding is generally thought interactions (see below). to occur independently of major influences outside this 2776 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein domain, we observed here a noteworthy effect on DNA more straightforwardly) by this unique biochemistry, binding by the presence or absence of the 6-amino-acid representing novel mechanisms for influencing genetic alternative insert (Hodgkinson et al. 1993) upstream of behavior. the basic region. Wild-type protein shows only a mod­ estly (20%) enhanced DNA affinity in the presence of Mi activates the pigmentation gene M-box element this insert, but a basic domain mutation (I212N, the One example of the biological activity of Mi was dem­ Mi^^ mutation; Steingrimsson et al. 1994) could be onstrated by its ability to trans-activate a reporter ele­ strikingly rescued for heterodimeric DNA binding by the ment driven by the M box (Fig. 5). This element contains insert (Fig. 4). This observation suggests that the 11 bp that are perfectly conserved in the promoters of the b-HLH-ZIP, and more specifically the 1212 site in the three major pigmentation enzyme genes in both mouse basic region, are subject to functionally important in­ and human and consists of 11 bp with a hexamer core of tramolecular interactions, an observation that may ex­ CATGTG (Shibahara et al. 1991; Lowings et al. 1992; tend to other b-HLH(-ZIP) factors. The location of the Yavuzer and Coding 1994). The immunoglobulin en­ 6-amino-acid insert, amino-terminal to the basic do­ hancer |JLE3 site contains the same core CATGTG and main, corresponds to the site of a 9-amino-acid alterna­ can be transcriptually activated by Mi (data not shown). tively spliced insert in Max (Blackwood and Eisenman It is attractive to speculate that through M-box recogni­ 1991). Kinetic data suggest that Max has a slower off rate tion. Mi provides a melanocyte-specific signal that acti­ and altered affinity in the presence of its 9-amino-acid vates the pigmentation program, potentially qualifying it insert (Bousset et al. 1993; Kretzner et al. 1993). Virtually as a master gene for melanocyte development. Although all b-HLH-ZIP proteins contain consensus casein kinase the M box can be bound by different b-HLH-ZIP pro­ II sites at this same location (see Fisher et al. 1993, and teins such as USF (Yavuzer and Coding 1994), Mi's trans- references therein). Phosphorylation appears to alter activation motif(s) might provide melanocyte-specific Max DNA binding in the direction of lower affinity (Ber- signals. This idea is consistent with the observation that berich and Cole 1992; Bousset et al. 1993), resulting in the M box is a melanocyte-specific enhancer element preferential heterodimeric DNA binding with Myc. The only when it is linked to the TATA box of a pigmenta­ presence and configuration of negatively charged moi­ tion gene promoter (Lowings et al. 1992). Therefore, even eties near the basic domain may influence protein-DNA if bound at an M-box site, different activator domains stability through repulsive forces with the DNA back­ might not function like that of Mi. Importantly, whereas bone. Similar influences of acidic residues upstream of Mi is expressed in a few tissues other than pigment cells, the basic domain of E12 significantly suppress ho- the alternative splice form in melanocytes appears to be modimeric DNA binding in this b-HLH factor (Sun and unique (Hodgkinson et al. 1993) and may represent a Baltimore 1991), suggesting that comparable mecha­ truly melanocyte-specific b-HLH-ZIP factor. It will be nisms operate in other basic domain-containing tran­ important to examine MiT family expression in cells scription factors. The b-HLH-ZIP protein USF contains affected by mi mutations. Two of Mi's dimerization part­ a direct repeat peptide sequence that resembles an im­ ners have been shown to encode transcriptional inhibi­ munoglobulin hinge motif (Gregor et al. 1990). The pres­ tory activity. TFEC represses TFE3-dependent transcrip­ ence of proline near the amino terminus of all b-HLH- tion (Zhao et al. 1993) and an alternative splice form of ZIP basic domains suggests that the peptide backbone is TFE3 has also been shown to repress the longer tran­ kinked in such a fashion that the upstream amino acids scriptionally active form of TFE3 (Beckmann et al. 1990; may reach back in the vicinity of the basic domain. It is Roman et al. 1991). Thus, regulated MiT protein dimers also interesting that the 1212 mutation (Steingrimsson et might direct the tissue-specific expression of pigmenta­ al. 1994) occurs on the solvent exposed surface of the tion program genes. basic domain. Although this position is not likely to con­ tact DNA (Fisher et al. 1993; Ferre-D'Amare 1993; Ste­ Mi also functions in melanocytes as a lineage-re­ ingrimsson et al. 1994), it is strikingly conserved as a stricted survival factor. During melanocyte develoment, hydrophobic residue in all CACGTG-binding b-HLH- cells harboring mi mutations appear to die, rather than ZIP proteins and is usually an arginine in CAGCTG (e.g.) survive without producing pigment. The prospect binding ones (Dang et al. 1992). Because the b-HLH-ZIP that pigmentation enzymes and melanocyte survival basic domain is an intrinsically unstable a-helix (Fisher genes are downstream effectors of Mi represents one of et al. 1993; Ferre-D'Amare et al. 1994), interactions on very few known transcription factor targets for the this other face may affect DNA binding by influencing b-HLH-ZIP family. An understanding of the role of Mi a-helical folding. Although the mechanism by which the in melanocyte development may provide insight into upstream region influences DNA binding remains un­ pathways of cellular proliferation and death in which clear, it is likely to be functionally important because of other b-HLH-ZIP proteins, like Myc/Max, are known to its biological consequences in mice carrying the mf^ or play roles. MT"^ mutations. The mild "enhancing" phenotype of mf^ lacking the insert (Wolfe and Coleman 1964) and Materials and methods the interallelic complementation of Mf^^ (Griineberg 1952; Hollander 1968; Konyukhov and Osipov 1968; DNA clones Steingrimsson et al. 1994) might both be explained [mi^^ The wild-type mi cDNA derived from melan-c cells was ex- GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. pressed in vitro from the clone pBS-Mi, which contains the ration from the initial (linear) slope of protein titrations under cDNA inserted into the EcoRl site of pBluescript SK -. This conditions of probe excess. Proteins were derived from in vitro cDNA lacks the 6-amino-acid alternative exon. Mutants corre­ translation reactions and were quantitated by determining sponding to the alleles mi (del 775-777), Mi°' (G776A), M^'' probe saturation in gel shift using probe of known specific ac­ (T764A), and the recessive alleles mi"^ (C916T), and mi"* tivity. Mi will aggregate with DNA in the absence of poly[d(I- C)]; therefore this nonspecific competitor was added to all re­ (G793A) (Steingrimsson et al. 1994) were generated by site-di­ rected mutagenesis of pBS-Mi using the method of Eckstein actions (as above). K^ measurements therefore reflect its pres­ according to the recommendations of the manufacturer (Amer- ence. Equilibrium conditions were established by incubation at sham). Templates for mi^"^, and constructs containing the 30°C for 75 min. Quantitation was carried out using a Phospho- 6-amino-acid alternative exon were expressed from PCR-de- rlmager (Molecular Dynamics). Immunoprecipitations were rived fragments made from wild-type as well as mi and Mi"^^ performed by mixing the various proteins under gel shift con­ mutant tissues. Expression templates were verified by DNA ditions (excluding poly [d(I-C)] and DNA probe) at 37°C for 1 hr, sequencing. TFEB was expressed from clone pTFEB-AA265 followed by addition of 3 [d of rabbit anti-TFEB antiserum and (Fisher et al. 1991). TFE3 in vitro expression vector was provided freshly washed protein A-Sepharose (Pharmacia), incubation at by Dr. T. Kadesch (Beckman et al. 1990). TFEC expression vec­ 4°C for 2 hr, and three washes with PBS containing 0.1% NP-40 prior to elution in loading buffer and SDS-PAGE. tor was provided by Dr. B. de Crombrugghe (Zhao et al. 1993). E47S was expressed from the plasmid pE47S (Murre et al. 1989). His fusion Mi was expressed from a plasmid containing the Transient transfections and luciferase assay BamHl-BamHl insert fragment from pBS-Mi inserted into the BamHl site of pET 15b (Novagen). For mammalian expression of NIH-3T3 cells were maintained in Dulbecco's modified Eagle Mi; the cDNA was cloned into the ffindlll and Xbal sites of medium supplemented with 5% calf serum/5% fetal calf se­ pRC-CMV (InVitrogen). The luciferase reporter plasmid was rum, 4 mM L-glutamine, 100 U/m l of penicillin, and 100 ixg/ml made by cloning an oligonucleotide containing four tandem re­ of streptomycin (GIBCO BRL). Cells were split 24—36 hr prior to peats of the M box (AGTCATGTGCT) into the Kpnl-Xhol sites transfection such that cells were ~60% confluent at the time of of the luciferase reporter plasmid pGL2 promoter (Promega). DNA addition, and were refed with fresh medium 8 hr prior to transfection. Transfections were carried out by calcium phos­ phate/DNA coprecipitation according to Kingston (1993) and Protein expression harvested after 24 hr. Three 6-cm plates were each transfected with 0.25 fjLg of luciferase reporter plasmid, 1 |xg of (3-galactosi- In vitro-translated proteins were made in rabbit reticulocyte dase control plasmid pRSV-p-Gal (Edlund et al. 1985), 4.7 jig of lysate (Promega) using RNA from in vitro transcription using cytomegalovirus (CMV)-driven expression vector pRC-CMV T7 RNA polymerase according to the manufacturer's recom­ (Invitrogen), and 4.05 |xg of carrier DNA pBS-SK (Stratagene). mendations (Pharmacia) for pBS-Mi and the corresponding mi, j^^wh^ j^^or^ j^^vit^ ^j ^-c e mutants as well as TFE3. Full-length At harvest, plates were washed once with phosphate-buffered Mi proteins were obtained by linearizing with Smal, and car- saline, lysed, and analyzed using a Monolight 2010 Luminom- boxy-terminal deletions at amino acids 319 and 261 were ob­ eter according to the recommendations of the manufacturer tained by linearizing with Xmnl and Avail, respectively. TFEB (Analytical Luminescence Laboratory, San Diego, CA). p-Galac- and E47S were transcribed using T3 RNA polymerase (Fisher et tosidase activity in cell lysates as a measure of relative trans­ al. 1991) (Pharmacia). Amino-terminal deletions and the DNA- fection efficiency was used to adjust luciferase data and was binding domain of TFEC were made by amplifying discrete frag­ assayed as described (Sambrook et al. 1989). ments using 5' primers that begin at the described residue and append an initiation ATG, Kozak sequence, and T3 RNA poly­ merase promoter (derived from the plasmid pBS-ATG, (Baldwin Acknowledgments et al. 1990) followed by transcription and translation in vitro. In We wish to thank Dr. Phillip Sharp for encouragement and sup­ vitro-translated proteins were quantitated by TCA precipitation port. Dr. Karen J. Moore for useful discussions, and Drs. T. and SDS-PAGE and equivalent quantities were added to gel shift Kadesch, B. deCrombrugghe and C. Miirre for plasmids. This assays. Recombinant TFEB was synthesized as described (Fisher work was supported in part by a grant from the Fimdacion In- et al. 1993). Recombinant His fusion Mi protein was synthe­ temacional Jose Carreras, and the National Cancer Institute sized in the bacterial strain BL-21, purified using nickel chelate under contract NOl-CO-74101 with ABL. chromatography (Qiagen), and eluted with 100 mM imidazole. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby Electiophoretic mobility shift assay, affinity measurements, marked "advertisement" in accordance with 18 USC section and immunoprecipitation 1734 solely to indicate this fact. DNA-binding assays were performed as described (Fisher et al. 1993) in 20-|xl reactions containing 5% glycerol, 100 mM KCl, References 10 mM Tris (pH 7.4), 1 mM DTT, and -5x10^^ cpm of ^^P-end- labeled probe DNA. In mixing experiments, separately trans­ Amati, B., M.W. Brooks, N. Levy, T.D. Littlewood, G.I. Evan, lated proteins were incubated at 37°C for 30 min prior to the and H. Land. 1993. Oncogenic activity of the c-Myc protein addition of probe DNA. CACGTG, CATGTG, and double point requires dimerization with Max. Cell 72: 233-245. mutant probes were used as described (Fisher et al. 1991). Poly- Anthony-Cahill, S.J., P.A. Benfield, R. Fairman, Z.R. Wasser- acrylamide gels (6% Tris-glycine-EDTA) were run and sub­ man, S.L. Brenner, W.F. Stafford, C. Altenbach, W.L. Hub- jected to autoradiography after drying. Competitors were pre­ bell, and W.F. DeGrado. 1992. Molecular characterization of pared as described previously (Fisher et al. 1991). Reactions helix-loop-helix peptides. Science 255: 979-983. probed with the CACGTG probe contained 1 jjig of poly[d(I-C)] Ayer, B. and R.N. Eisenman. 1993. A switch from Myc:Max to per 20-|xl reaction, whereas those containing CATGTG probe Mad:Max heterocomplexes accompanies monocyte/macro­ contained 0.5 |xg. K^ was determined by calculating half satu­ phage differentiation. 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Yavuzer, U. and C.R. Coding. 1994. Melanocyte-specific gene expression: Role of repression and identification of a mela­ nocyte-specific factor, MSF. Mol. Cell. Biol. 14: 3494-3503. Zervos, A.S. 1993. Mxil, a protein that specifically interacts with Max to bind Myc-Max recognition sites. CeU 72: 223 - Zhao, G.-Q., Q. Zhao, X. Shou, M.-G. Mattel, and B. DeCrom- brugghe. 1993. TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent tran­ scription activation. Mol. Cell. Biol. 13: 4505-4512. 2780 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. T J Hemesath, E Steingrímsson, G McGill, et al. Genes Dev. 1994, 8: Access the most recent version at doi:10.1101/gad.8.22.2770 This article cites 52 articles, 27 of which can be accessed free at: References http://genesdev.cshlp.org/content/8/22/2770.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Copyright © Cold Spring Harbor Laboratory Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes & Development Unpaywall

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Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family Timothy J. Hemesath / Eirikur Steingrimsson,^ Gael McGill / Michael J. Hansen / James Vaught/ Colin A. Hodgkinson,^ Heinz Amheiter,^ Neal G. Copeland/ Nancy A. Jenkins/ and David E. Fisher^'* ^Division of Pediatric Hematology/Oncology, Dana Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 USA; ^Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 USA; ^Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 USA The ndcrophtbalinia (mi) gene appeals essential foi pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities. [Key Words: microphthalmia; dimerization; DNA binding; M box; b-HLH-ZIP] Received August 23, 1994; accepted in revised form October 11, 1994. of melanocyte growth or survival. The mi gene was A striking inheritable disorder of development in the cloned recently and shown to predict a basic/helix- mouse is microphthalmia [mi], a syndrome first recog­ loop-helix/leucine zipper (b-HLH-ZIP) factor (Hodgkin- nized >50 years ago as a coat color mutation (Hertwig son et al. 1993; Hughes et al. 1993; Tachibana et al. 1942). The human mi gene has also been linked compel- 1994). Mammahan b-HLH-ZIP factors and the related lingly to the human pigment cell disorder Waardenburg b-HLH group contain several important regulators of Syndrome (Hughes et al. 1994). Mutations at the mouse cell proliferation and development such as Myc/Max mi locus result in pigment cell defects in the skin (pro­ (Blackwood and Eisenman 1991; Prendergast et al. 1991) ducing white spotting), eyes (producing small eyes), and and MyoD-related factors (for review, see Olson 1990, inner ears (resulting in deafness). Mast cell defects have and references therein; Weintraub 1994). Given the ef­ also been recognized for certain mi alleles, a pattern re­ fects of mi mutations on melanocyte biology, mi may sembling the melanocyte/mast cell pattern of SI/kit-de­ regulate comparable pathways in melanocytes. Although fective mice and suggesting a coimection between these a significant number of b-HLH-ZIP factors are known, factors in signaling (Dubreuil et al. 1991; Ebi et al. 1992). biological activities are clear for only a few, primarily Bone resorption and other neural crest or neuroepithelial defects have also been observed for certain mi alleles (for Myc and its related partners (see Prendergast and Ziff review, see Green 1989), suggesting that Mi protein may 1992; Ayer and Eiseimian 1993; Zervos 1993). Only very function in part through oligomeric interactions with few candidate target genes have been identified so far other factors. that are regulated by these factors. The striking biologi­ cal consequences of mi mutations suggest a major role in The devastating consequences of mi mutations on me­ melanocyte development and even point to specific can­ lanocyte development suggest that Mi is a key regulator didate target genes. Melanocytes represent a neural crest-derived lineage *Conesponding author. whose pigmentation function is easily assessed because GENES & DEVELOPMENT 8:2770-2780 © 1994 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/94 $5.00 2770 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein and protein-DNA interactions have been revealed melanocytes are not critical to survival of the whole an­ through a characterization of the proteins encoded by imal. A great deal has been learned about pigmentation seven mutant mi mouse alleles (Steingrimsson et al. enzymes and their regulation through study, for exam­ 1994). These mutations cluster within or near the ple, of albino (tyrosinase) mutants (for review, see Hala- b-HLH-ZIP motif of mi and display striking effects on ban and Moellmann 1993). Determination of regulatory heterodimerization and DNA binding that largely ex­ elements critical for pigmentation have revealed an 11- plain the unique severity and inheritance patterns of the bp sequence known as the M box containing the core different mi mouse strains. Novel structural features of element CATGTG, which is highly conserved in the pro­ b-HLH-ZIP biochemistry have also been revealed, such moters of the three major pigmentation enzyme genes as the surprising ability of an alternative exon outside of tyrosinase, and tyrosinase-related proteins 1 and 2 the basic domain to modulate b-HLH-ZIP-dependent (Shibahara et al. 1991; Lowings et al. 1992; Yavuzer and DNA recognition. Finally, Mi was shown to transcrip­ Coding 1994). The presence of tissue-specific transcrip­ tionally activate a reporter driven by the pigmentation tion factors capable of interacting with and transcrip­ promoter M-box element, suggesting that this family of tionally activating elements such as this may shed light factors plays a central role in the tissue-specific devel­ on the regulation of pigmentation and perhaps other me- opment of melanocytes. lanocyte-specific functions. The opportunity to examine structure/function rela­ tionships for transcription factor mutations coordinately in vitro and in mice has rarely been possible. The mul­ Results titude of mi mouse mutants provides a unique opportu­ DNA recognition by Mi nity to examine biochemical consequences of biologi­ cally important b-HLH-ZIP mutations. The b-HLH- The Mi protein produces a gel shift complex (Fig. 1) with ZIP family contains a short ~20-amino-acid basic DNA containing the CACGTG hexanucleotide derived domain rich in basic amino acids that makes sequence- from adenovirus major late promoter (MLP). Several de­ specific DNA contacts. Carboxy-terminal to it is the letions were made to determine the protein domains re­ HLH-ZIP containing two amphipathic helices separated quired for DNA binding (Fig. 1 A). It was possible to trun­ by a flexible loop and a carboxy-terminal leucine zipper. cate from the amino terminus to the beginning of the The HLH-ZIP mediates dimeric interactions necessary basic domain and from the carboxyl terminus to the end for DNA binding (Ferre-D'Amare et al. 1993). Restricted of the leucine zipper domain without loss of DNA bind­ heterodimerization plays a major role in regulating de­ ing (Fig. IB, lanes 2-4). Further deletion from the car­ velopmental programs ranging from inhibition of myo- boxyl terminus removed part of the leucine zipper and genesis by the HLH protein Id (Benezra et al. 1990) or abolished DNA binding (Fig. IB, lane 5). Therefore, the morphogenesis by extiamachiocaete (Ellis et al. 1990; leucine zipper was essential for stable complex forma­ Garrel and Modolell 1990) to cooperation in cellular tion. transformation by Myc/Max (Blackwood and Eisenman Sequence specificity of DNA recognition was verified 1991; Prendergast et al. 1991; Kato et al. 1992; Amati et by competition analysis using both CACGTG (Fig. IC) al. 1993). The ability to group these factors into families, and CATGTG (Fig. ID) probes. In each case, a double based on dimerization specificities, provides a useful point mutant (GAGGTG) failed to compete the specific handle for analysis of their biological roles. complex at concentrations effectively competed by un­ Most b-HLH-ZIP proteins recognize the hexamer core labeled CACGTG competitor. sequence CACGTG or the related sequence CATGTG, whereas AP-4 (Hu et al. 1990) and most b-HLH proteins recognize CAGNTG hexamers. A hexamer containing Mi is a member of a discrete family of b-HLH-ZIP the CATGTG sequence is present in the mouse immu­ factors noglobulin heavy chain enhancer and was used to isolate and characterize the transcription factor TFE3 (Beck- Stoichiometry of protein to DNA in the bound com­ maim et al. 1990; Roman et al. 1992). Although most plexes was examined by mixing full-length Mi protein b-HLH-ZIP factors interact avidly with cognate targets, with the isolated b-HLH-ZIP region (Fig. 2, lanes 2 and it has been difficult to elucidate tissue-specific activities, 3, respectively). A single new intermediate mobility gel in part because most of these factors are expressed ubiq­ shift complex was observed (Fig. 2, lane 4). Overexposure uitously. In this regard, mi, which is tissue restricted, is failed to reveal additional intermediate complexes, sug­ an attractive candidate as an M-box activator and regu­ gesting that the protein-DNA stoichiometry is 2:1. lator of pigmentation gene expression. Experiments were also imdertaken to determine The studies described here identify Mi's DNA-binding whether Mi is capable of forming DNA-binding het- activity and its ability to form stable DNA-binding het- erodimers with several other b-HLH-ZIP proteins. Only erodimers with TFEB, TFE3, and TFEC, three other three proteins, TFEB, TFE3, and TFEC, were found to b-HLH-ZIP factors. Collectively, these four proteins form intermediate mobility complexes with Mi (Fig. 2, comprise a distinct family that likely modulates the bi­ lanes 5-13). In these mixing experiments TFE3 (but not ological activity of Mi through hetero-oligomer forma­ Mi) preferentially heterodimerizes, probably reflecting tion. The biological importance of Mi's protein-protein different kinetics from Mi. In contrast, no heterodimers GENES & DEVELOPMENT 2771 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. A alt I .. V exo . n bHLHZIP 419 aa NA192 OO CD CA261 CA308 S < O Competitor: GAGGTG CACGTG CA319 < ^, Ol CJ CL o o 2 = ?5 °2 N (ng) o o o o ■> — T — CC 3 <i <] o5 i n CM i n (N D Competitor: CACGTG GAGGTG ■o UL 6 (ng) 1 10 30 50 1 10 30 50 » 1- CATGTG Free probe CACGTG DNA probe 1 2 3 4 5 6 7 8 1 2 3 4 5 4 5 6 1 2 3 Figure 1. mi gene and deletion analysis. (A) Schematic representation of the Mi protein. Hatched areas depict basic helix-loop-helix, and leucine zipper domains (b-HLH-ZIP). Positions of the various amino-terminal and carboxy-terminal deletions are indicated using numbering based on the melanocyte form of Mi that contains 419 amino acids (Hodgkinson et al. 1993; Steingrimsson et al. 1994). {B) DNA binding by Mi. Radiolabeled DNA containing the core hexanucleotide CACGTG was used in gel shift analysis. Proteins were derived from in vitro translation using reticulocyte lysate and were either full length or truncated as indicated. Endogenous reticu­ locyte DNA binding is indicated by an asterisk (*).(- RNA) Reticulocyte lysate unprogrammed by exogenous RNA. (C) Competition for CACGTG probe. Mi protein containing an intact b-HLH-ZIP region (truncation NA192;CA308) was tested for DNA binding to radiolabeled CACGTG probe. Unlabeled competitor DNA fragments of identical size were added as indicated in units of nanograms. Core sequences of the competitors are shown. (*) A background reticulocyte activity; {—>■] Mi specific activity. [D] Competition for CATGTG probe. Purified recombinant Mi protein was bound to radiolabeled |J.E3 probe DNA in the presence of unlabeled competitor DNAs as indicated, (^►j The Mi specific activity; (*) a contaminating activity present in the probe. were observed upon mixing Mi with E47S (Fig. 2, lanes produced and tested directly for their ability to bind 14-16), Max, Myc, upstream stimulatory factor (USF), or DN A as homodimers or as heterodimers with TFE3. several non-HLH-containing transcription factors (data Identical result s wer e obtained whe n heterodimerization no t shown). Therefore, of the known and tested candi­ was tested with wild-type Mi, TFEB, or TFEC (data not date partners. Mi appears to be capable of forming stable shown). The seven mutations and their properties are DNA-bindin g heterodimers wit h only TFEB, TFEC, and summarize d in Table 1. TFE3. With the additional observation that TFEB and Whe n tested for homodimeric DN A binding, all three TFEC form stable heterodimer s (Fig. 2), all combinations semidominan t and two recessive mutan t proteins failed of these four proteins have now been shown to het- to bind DN A (Fig. 3A, lanes 3-8). Only the helix 1 mu­ erodimerize with one another but not with any other tan t D222N (mi^") mutan t protein, which is inherited know n b-HLH-ZIP proteins (Fig. 2; Fisher et al. 1991; recessively, appeared to bind DN A normally. Quantita­ Zha o et al. 1993), indicating that they constitute a dis­ tive affinity measurements revealed mi'' " t o bind wit h a crete group of interactive proteins, which we refer to as K^ only 6% greater than that of wild-type Mi (using th e MiT family. forms containing the 6-residue alternative insert), a dif­ ference within the 10% standard error of our measure­ ment s (data not shown). When mixed with TFE3, mi'*'" Mutant alleles affect MiT interactions wa s the only mutant protein able to produce a het- Recen t molecular genetic studies of Steingrimsson et al. erodimeric complex wit h TFE3 (Fig. 3A, lanes 10-16). (1994) suggest tha t dominant-negative Mi mutation s are Examinatio n of heterodimer mixing experiments us­ dominantl y inherited whil e regulatory mutation s or mu­ ing th e mutan t Mi proteins (Fig. 3A) revealed a striking tation s that prevent or reduce Mi protein dimerization loss of TFE3 homodime r activity in man y reactions. Ad­ are recessively inherited. To examine this possibility, dition of mi. Mi""", Mi"^^, or mi^^ proteins essentially mutan t proteins corresponding to the seven mi muta­ ablated TFE3 homodimeric DNA-binding activity (Fig. tion s characterized by Steingrimsson et al. (1994) were 3A, lanes 12,13,15,16). In contrast, the recessive allele GENES &, DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein < Mi/Mi TFEB/Mi TFE3/Mi Mi/TFEC E47/Mi TFEC/TFEB protein-protei n interactions, coimmunoprecipitations were performed using ^^S-labeled mutant Mi proteins, '^IM B TMB TMB MT B EiB TcTbB unlabeled recombinant TFEB, and a TFEB-specific anti­ mt'-mM *"»"""'wi iP*'W ..|«||| ^^^^ body. Antibod y specificity was verified by supershift of a TFEB/DN A complex but failure to supershift Mi or ■ " ■ • ■■ ■ ^" fMj othe r b-HLH-ZI P proteins (data not shown). Specificity wa s indicated further by the dependence for TFEB i n the Mi Mi HI Mi Mi V' I coimmunoprecipitation s (Fig. 3B, lanes 1,2) as well as th e dependence of antibody (data not shown). The la­ beled Mi protein migrates as a doublet of —15 Kd. TFEB- specific antibodies coimmunoprecipitated wild-type Mi, th e three semidominant proteins mi, Mi°'', and Mi"^"^, as wel l as the recessive protein mi'*''* (Fig. 3B, lanes 2-6), consisten t with a dominant-negative inhibition of DN A 1 2 3 4 5 6 7 8 9 10 11 12 13 141516 17 18 19 binding by the products of the semidominant alleles. A Figure 2. Mi forms stable heterodimers with TFEB, TFE3, and similar coimmunoprecipitation pattern was also ob­ TFEC. Full-length and truncated forms of various b-HLH- served for mi^'^ (data no t shown). Th e zipperless reces­ ZIP proteins were translated separately in vitro and equivalent sive protein mi''^ did not efficiently coprecipitate (Fig. volumes were mixed (post-translationally) prior to the addi­ 3B, lane 7), althoug h a weak signal was observed, possi­ tion of radiolabeled CACGTG probe. (M) The Mi truncation bly reflecting a propensity to form HLH-mediated tet- NA192;CA308; (Mi) full-length Mi protein; (B) both proteins ramer s in the absence of DN A (Fisher et al. 1991; An- mixed together; (T) TFEB, TFE3, or TFEC as indicated; (Tc) thony-Cahil l et al. 1992; Farmer et al. 1992; Fairman et TFEC; (Tb) TFEB. The Mi used in lanes 3-10 encompasses the b-HLH-ZIP region; the Mi used in lanes 2 and 11-16 is full al. 1993). length. The fragment of TFEB used contains all but the first 265 amino acids (TFEB-AA265; Fisher et al. 1991). TFE3 is full- length (Beckmaim et al. 1990). The TFEC fragment contains the Alternative splice affects basic domain function isolated b-HLH-ZIP region (NA99;CA204). E47S is a truncation Th e mi message exists in splice forms either encoding or of E47 that includes the b-HLH region (Miirre et al. 1989). lacking 6 amino acids just amino-terminal to the basic domai n (Hodgkinson et al. 1993). Th e mf^ mutation af­ fects the polypyrimidine tract of the splice acceptor and mi''^ contains a stop codon tha t removes the leucine zip­ precludes formation of Mi protein containing the per and did no t affect TFE3-binding activity (Fig. 3A, lane 6-amino-acid insert (Steingrimsson et al. 1994). These 11). All three semidominant alleles contain basic do­ mic e produce normal pigment but exhibit a measurable mai n mutations, failed to bind as homodimers, and ad­ decrease in the pigmentation enzyme tyrosinase within ditionally suppressed DN A binding by TFE3 in an appar­ ski n (Wolfe and Coleman 1964). Despite the subtlety of entl y dominant-negative fashion. it s homozygous phenotype, the mi^^ allele enhances the effective phenotype of semidominant mi alleles in a Surprisingly, of the three recessive mutant proteins, compoun d heterozygote (Wolfe and Coleman 1964). To mi'' " bound DN A indistinguishably from wild-type pro­ examin e biochemical relevance of thi s alternative splice, tei n despite its helix 1 mutatio n (and the striking phe- wild-type Mi proteins with and without the 6-amino- notyp e of mi^^ mice) (Fig. 3A, lanes 6,14), suggesting acid insert were examined (Fig. 4A, lanes 2,3). Although tha t this mutation might disrupt a function other than th e two proteins boimd DNA similarly, quantitative DN A binding. Th e recessive allele, mi''^, contains a stop measurement s revealed that the splice form containing codon at the begiiming of the leucine zipper, failed to th e insert bound wit h 20% higher affinity than the form bind DNA, and was also incapable of suppressing the lacking th e insert (K^ = 290 and 349 JJLM, respectively , in DNA-bindin g activity of TFE3 (Fig. 3A, lanes 3,11) be­ presence of poly [d(I-C)]. N o large effect was observed for havin g "recessively" in vitro. A third recessive muta­ th e alternative 6-amino-acid insert on heterodimeric tion, mi^^, contains a 25-amino-acid deletion that re­ binding of wild-type Mi wit h TFE3 (Fig. 4A, lanes 4—6). move s the amino-terminal half of the basic region but Surprisingly, however, the presence of the 6-amino- does not involve the HLH-ZIP. This mutant failed to acid insert had a profound effect on DN A binding of the bind DNA as a homodimer and also suppressed the basic domain mutan t I212N (Mi^"^), th e allele that dis­ DNA-bindin g activity of TFE3 (Fig. 3A, lanes 8,16). As a basic domain deletion, this in vitro behavior was ex­ plays interallelic complementation. As shown in Figure pected for mi^^. Its recessive inheritance is surprising, 4B, presence of the insert restored heterodimeric DNA however. Importantly, this discrepancy between th e bio­ binding by Mi^'^ wit h a wild-type partner (Fig. 4B, lanes chemica l behavior of the mi^"^ protein and the genetic 1-4,9,11). In contrast, presence of the upstream insert behavior of th e mi^"^ allele suggests tha t these deleted 25 did not restore heterodimeric DN A binding for a differ­ en t basic region mutan t (mi), indicatin g th e specificity of amin o acids carry out a second function (aside from thi s effect for Mi"^^ (Fig. 4B, lanes 5-7,10). Thus, pres­ DN A binding). ence of th e upstrea m insert restored DN A binding to the To verify that the TFE3 suppression seen by proteins jyjjwh protein if the heterodimer partner was wild type. encoded by the semidominant alleles occurred through GENES & DEVELOPMENT 2773 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. Table 1. Miciophthalmia mutant alleles DNA binding Symbol Mutation^ Description homo- hetero-^ dom-neg*^ Semidominant microphthalmia mi no no yes del R217'* deletion within basic domain oak ridge Mi°' R216K no no yes basic domain mutation (facing DNA) white Mr^ I212N basic domain mutation (away from DNA) no yes^ yes* Recessive cloudy eyed no no no mf^ R263 STOP deletes leucine zipper and carboxyl terminus eyeless white mf"^ del A187-I212 deletion into basic domain no no yes vitiligo mr' D222N helix 1 mutation yes yes no Enhancing spotted mfP del 186-191 loss of alternative 6-amino-acid exon yes yes no Interallelic complementation white Mr'' I212N basic domain mutation (away from DNA) no yes* yes* ^Steingrimsson et al. (1994). ''Heterodimeric DNA binding tested with wild-type binding partners (TFE3 and Mi). '^Dominant-negative effects tested through inhibition of homodimeric wild-type protein in same reaction. ■^The mi allele deletes an Arg codon among a cluster of four in the basic domain. It is unclear which one has been deleted. e^^jwh mutant protein can bind as heterodimer with wild type only in presence of the 6-amino-acid upstream insert and suppresses (dom-neg) only in absence of insert. fjVlj«''i mutant protein is dominant negative only in the absence of the 6-amino-acid upstream insert. suggesting that this 6-amino-acid insert acts to stabihze family of proteins and whose complexity of allelic in­ the basic domain/DNA complex, hiterestingly, the teractions may be largely explained by these features. I212N mutation in the Mi"^*" protein is the only basic Biochemical analysis of Mi demonstrated its capacity to region mutant predicted to face away from DNA in the specifically recognize the DNA core sequences basic domain a-helix, on the solvent-exposed face (Ferre- CACCTC and CATGTG (Fig. 1). This DNA binding ap­ D'Amare et al. 1993; Fisher et al. 1993; Steingrimsson et peared to be dimeric based on mixing experiments that al. 1994). The restoration of DNA binding for Mi"^*" may result in the formation of a single intermediate mobility account for the interallelic complementation character­ complex (Fig. 2). Although this observation does not for­ istic of this allele. mally prove 2:1 stoichiometry of protein to DNA, the DNA cocrystallographic analyses of Max and USF showed dimeric protein interaction with the cognate Mi over expression tianscriptionally activates DNA template (Ferre-D'Amare et al. 1993, 1994). Addi­ an M box-driven reporter in fibroblasts tionally, the importance of Mi's leucine zipper was dem­ onstrated by the loss of DNA binding upon its deletion. We have tested the ability of mi to activate transcription A substantial body of data indicate that the leucine zip­ of a reporter driven by the M-box pigmentation gene pro­ per is necessary for dimerization and DNA binding by moter element (Shibahara et al. 1991; Lowings et al. b-HLH-ZIP proteins (Dang et al. 1989; Gregor et al. 1992; Yavuzer and Coding 1994) because of our demon­ 1990; Beckmaim and Kadesch 1991; Blackwood and stration that Mi is capable of binding its CATGTG core Eisenman 1991; Fisher et al. 1991; Prendergast et al. sequence in vitro (Fig. ID). Cotransfection of mi and the 1991; Blanar and Rutter 1992; Roman et al. 1992). M-box reporter into NIH-3T3 cells resulted in Mi-depen­ dent activation of the luciferase gene to levels > 13-fold above controls (Fig. 5). Stimulation of the luciferase ac­ Mi belongs to a discrete MiT family tivity was dependent on both the presence of the M-box element in the reporter construct and on the cotransfec­ Based on the phenotypic complexity of heterozygous tion of mi. Although identical to the immunoglobulin combinations of mi alleles (for review, see Green 1989), enhancer element |xE3 element at its core (CATGTG), it is likely that mi function depends on heterodimer for­ the M box differs in flanking positions, which are con­ mation during development. Heterodimeric DNA bind­ served from mouse to human in the three pigmentation ing was seen for Mi protein in combination with TFEB, TFE3, or TFEC (Fig. 2). With the observation that TFEB enzyme genes tyrosinase, and tyrosinase-related proteins and TFEC were also capable of heterodimerization and 1 and 2. Recognition of M-box elements by Mi may con­ DNA binding, all dimeric combinations of these factors stitute a critical component in the elaboration of mela- have now been demonstrated (Fig. 2; Fisher et al. 1991; nocyte-specific gene expression. Zhao et al. 1993). Heteromeric DNA-binding interac­ tions are otherwise quite restricted for these proteins, as Discussion none of them have been shown to heterodimerize with other HLH or HLH-ZIP factors. Whereas TFEB and TFE3 The experiments presented here demonstrate that the are ubiquitous factors (Beckmann et al. 1990; Carr and Mi protein is a transcription factor that forms homo- and Sharp 1990) and TFEC is tissue restricted (Zhao et al. heterodimeric DNA-binding complexes within a small 2774 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmi a protein importan t in the human pigmentation disorder Waar- denburg Syndrome^ which is dominantly inherited and ^ > LU I— was recently linked to th e huma n mi locus (Hughes et al. 1994). Th e behavior of these mutants , particularly those wit h unanticipated protein function, may aid in the ^migmm^S TFE3+ + LU h- I- hr h- I- U_ D C ':''&f. '_ wK~W^ 1 2345678 9 1011 1213U151 6 1 l-tb. - + + + + + + 1 2 3 4 5 6 TFE3 + TFE3 + ro + + 9HMMBI^$^^'^^ ^ : a: 14.3 ■fi t \^ ■§ s S ■f 5 £ 1 2 3 4 5 6 7 ¥" 3 Figure 3. DNA-binding properties of mi mutants. (4) Wild- type Mi (Mi-WT) and six different mutant Mi proteins were «?«» synthesized in vitro as amino-terminal deletions beginning with amino acid 109 (to visualize intermediate mobility com­ plexes). Proteins were tested in DNA-binding assays either alone (lanes 2-8] or in post-translational mixes with TFE3 (lanes 10-16]. The semidominant alleles mi, Mi°'', Mi^^, and the re­ cessive alleles mi"'', mi"\ and mi^^ were tested. The positions of two background reticulocyte bands are indicated (*), the lower one being remote from the strong signals and demonstrat­ ing evenness of sample loading, [B] Immunoprecipitation of wild-type (WT) and mutant Mi proteins with unlabeled recom­ 9 10 n 12 1 2 3 5 6 7 binant TFEB, using a TFEB specific antibody. Specificity is seen in lane 1, where lack of TFEB results in no coprecipitation. Figure 4. Alternative splice restores heteromeric DNA binding Wild-type Mi, the three dominant-negative proteins (mi, Mi°', by Mi'"'*. [A] DNA binding by two splice forms of Mi. Wild-type and Mi^*^), and mi"'^ coprecipitate efficiently with TFEB (lanes Mi protein (AA109;CA308) either lacking (WT - ) or containing 2-6); mi*^^, a zipperless protein, is very weakly coprecipitated, (WT +) the 6-amino-acid alternative exon was tested for DNA perhaps through a propensity to form HLH-dependent tetramers binding using the CACGTG probe, either alone or in the pres­ (lane 7). ence of TFE3. No obvious differences in DNA binding or het- erodimerization were apparent. Several background bands (*) represent reticulocyte proteins capable of DNA binding. [B] Six- 1993), i t wil l be importan t to determin e th e developmen­ amino-acid insert restores heterodimeric DNA binding by Mi^^. The basic domain mutant Mi*"" (AA109;CA308) was syn­ tal expression of these factors within cell lineages af­ thesized either with (Wh -I- j or without (Wh - ) the 6-amino-acid fected by mi mutations. Thus, these four proteins repre­ insert and tested for DNA binding (CACGTG) in the presence of sen t a distinc t Mi T family that likely participates in piv­ TFE3 (lanes 1-8] or alone (lane 9). A truncated form of Mi^'' otal developmental pathways, although other family contains only the b-HLH-ZIP (Wh-b). Another basic domain member s might exist as well. mutant, mi, was also synthesized from amino acid 109 (AA109;CA308) in the presence {mi + ] or absence (see Fig. 3) of the 6-amino-acid insert and tested for DNA binding with TFE3 Biochemical lesions and biological consequences (lane 6) or alone (lane 10]. Presence of the 6 amino acids restored We show here that dominant-negative protein behavior heterodimeric DNA binding to the Mi"^^ mutant (—>) without affecting the mi protein. Several background reticulocyte lysate appears to explain semidominant inheritance of mi alle­ bands are observed (see lane 12, unprogrammed lysate). les. This is relevant for mouse mi and is likely to be GENE S & DEVELOPMEN T 2775 Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. Mi proteins producing recessive inheritance are also instructive regarding b-HLH-ZIP function and highlight functionally relevant regions unlikely to produce the S 150 dominant inheritance of Waardenburg Syndrome. Impor­ tantly, two (mi'^^ and mi^^] of the three display bio­ chemical behavior that is not expected. The third, mi'^^, introduces a stop codon at the carboxyl terminus of the HLH domain (Steingrimsson et al. 1994), thereby trun­ cating the leucine zipper. The transcription factor USF, however, appears to be capable of binding DNA without % 50 its leucine zipper (Gregor et al. 1990; Ferre-D'Amare et al. 1994). By failing to dimerize, the mi''^ protein should exert no dominant-negative effect at the level of DNA binding, as was observed in mixing experiments (Fig. 3). Luc + M-Luc + Luc + M-Luc + The weak coimmunoprecipitation of mi*^® by TFEB (Fig. vector vector Mi Mi 4) suggests that the HLH domain alone can measurably Figure 5. Mi stimulates transcription from a promoter con­ oligomerize, perhaps as a tetramer, in the absence of struct containing M-box elements. NIH-3T3 cells were tran­ DNA (Anthony-Cahill et al. 1992; Farmer et al. 1992; siently transfected and assayed after 24 hr for luciferase activity Fairman et al. 1993; Fisher et al. 1993). (expressed in relative light units). Error bars represent the stan­ dard deviation of triplicate samples. Transfected DNA con­ The mf^ allele predicts a 25-amino-acid deletion tained a luciferase reporter plasmid containing a minimal SV40 (Steingrimsson et al. 1994) that begins amino-terminal to promoter alone (Luc) or carrying four upstream copies of an (and deletes much of) the basic domain. This protein M-box element (M-Luc), and a CMV-driven expression vector failed to bind DNA as either a homodimer or heterodi- alone (vector) or containing a cDNA encoding wild-type Mi (Mi) mer. Like the semidominant alleles, it repressed DNA lacking the 6-amino-acid alternative insert. Weak M box-spe­ binding by wild-type protein because the HLH-ZIP do­ cific basal activity is seen in 3T3 cells as well as strong Mi- mains were intact. Interestingly, the mi^^ allele is in­ specific trans-activation. herited recessively suggesting that dominant-negative function is not fully realized in vivo. Potential explana­ identification and characterization of human mi lesions tions include the loss of a nuclear localization signal or capable of producing Waardenburg SyndromC; eventually decrease in protein stability. allowing for genetic screening in affected families. DNA The D222N mutations [mi^^] produces a helix 1 mu­ recognition by the basic domain can be disrupted in sev­ tation (Steingrimsson et al. 1994) with virtually no mea­ eral ways, some of which are reminiscent of the MyoD surable effect on DNA binding (Fig. 3) but produces pro­ inhibitor Id (Benezra et al. 1990) and the Diosophila fac­ gressive, aging-dependent melanocyte death (Lemer tor extiamacwchaete (Ellis et al. 1990; Garrel and Mo- 1986; Lemer et al. 1986). It is possible that the small dolell 1990). (6%) difference in K^ produced by this mutation is suf­ ficient to produce the aging-dependent vitiligo in these The basic domain of b-HLH-ZIP proteins recognizes mice. Altematively, this helix 1 mutation may affect DNA through a discrete a-helical face (Fisher et al. 1991) tetramerization, a property of many HLH proteins. TFEB that forms an iminterrupted structure with helix 1 of the has been shown previously to exist in a tetrameric state HLH domain (Ferre-D'Amare et al. 1993). This is an in­ in solution that dissociates into DNA-binding dimers trinsically unstable a-helix requiring DNA binding to upon addition of DNA (Fisher et al. 1991). Similar tet- stabilize its folding (Fisher et al. 1993; Ferre-D'Amare et ramers have been observed for several other HLH-con- al. 1994). The mi protein lacks a basic region arginine taining proteins including Myc (Dang et al. 1989), MyoD (Hodgkinson et al. 1993) which should shift the rota­ (Anthony-Cahill et al. 1992), and myogenin (Farmer et al. tional register of the basic domain a helix by —100° rel­ 1992). The Id protein's inhibition of MyoD DNA binding ative to the HLH, precluding DNA binding. The R215K appears to be mediated by tetrameric complexes (Fair- mutation in Mi°'' (Steingrimsson et al. 1994) destroys man et al. 1993) consistent with the observation that DNA binding in TFEB (Fisher et al. 1993) as well as in Mi tetrameric forms carmot bind DNA (Fisher et al. 1991). (Fig. 3). Although this position appeared to only make a The aspartate 222 mutated in mi"^* (Steingrimsson et al. phosphate contact in the cocrystal structure of Max/ 1994) is located within the four-helix bundle predicted DNA (Ferre-D'Amare et al. 1993), the fact that lysine could not substitute suggests another critical fimction, from the Max/DNA cocrystal structure (Ferre-D'Amare most likely including salt bridge formation with the up­ et al. 1993) and could participate in interhelical salt stream glutamate, thereby stabilizing a-helical folding. bridges, although its disruption does not appreciably af­ Surprisingly the semidominant mutation I212N {Mi^^] fect dimerization. is predicted to face away from the major groove of the DNA on the basic domain a-helix (Fisher et al. 1991, Alternative splice modulates DNA binding 1993; Ferre-D'Amare et al. 1993) and provides evidence that the basic domain is subject to significant regulatory Although b-HLH-ZIP DNA binding is generally thought interactions (see below). to occur independently of major influences outside this 2776 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Microphthalmia protein domain, we observed here a noteworthy effect on DNA more straightforwardly) by this unique biochemistry, binding by the presence or absence of the 6-amino-acid representing novel mechanisms for influencing genetic alternative insert (Hodgkinson et al. 1993) upstream of behavior. the basic region. Wild-type protein shows only a mod­ estly (20%) enhanced DNA affinity in the presence of Mi activates the pigmentation gene M-box element this insert, but a basic domain mutation (I212N, the One example of the biological activity of Mi was dem­ Mi^^ mutation; Steingrimsson et al. 1994) could be onstrated by its ability to trans-activate a reporter ele­ strikingly rescued for heterodimeric DNA binding by the ment driven by the M box (Fig. 5). This element contains insert (Fig. 4). This observation suggests that the 11 bp that are perfectly conserved in the promoters of the b-HLH-ZIP, and more specifically the 1212 site in the three major pigmentation enzyme genes in both mouse basic region, are subject to functionally important in­ and human and consists of 11 bp with a hexamer core of tramolecular interactions, an observation that may ex­ CATGTG (Shibahara et al. 1991; Lowings et al. 1992; tend to other b-HLH(-ZIP) factors. The location of the Yavuzer and Coding 1994). The immunoglobulin en­ 6-amino-acid insert, amino-terminal to the basic do­ hancer |JLE3 site contains the same core CATGTG and main, corresponds to the site of a 9-amino-acid alterna­ can be transcriptually activated by Mi (data not shown). tively spliced insert in Max (Blackwood and Eisenman It is attractive to speculate that through M-box recogni­ 1991). Kinetic data suggest that Max has a slower off rate tion. Mi provides a melanocyte-specific signal that acti­ and altered affinity in the presence of its 9-amino-acid vates the pigmentation program, potentially qualifying it insert (Bousset et al. 1993; Kretzner et al. 1993). Virtually as a master gene for melanocyte development. Although all b-HLH-ZIP proteins contain consensus casein kinase the M box can be bound by different b-HLH-ZIP pro­ II sites at this same location (see Fisher et al. 1993, and teins such as USF (Yavuzer and Coding 1994), Mi's trans- references therein). Phosphorylation appears to alter activation motif(s) might provide melanocyte-specific Max DNA binding in the direction of lower affinity (Ber- signals. This idea is consistent with the observation that berich and Cole 1992; Bousset et al. 1993), resulting in the M box is a melanocyte-specific enhancer element preferential heterodimeric DNA binding with Myc. The only when it is linked to the TATA box of a pigmenta­ presence and configuration of negatively charged moi­ tion gene promoter (Lowings et al. 1992). Therefore, even eties near the basic domain may influence protein-DNA if bound at an M-box site, different activator domains stability through repulsive forces with the DNA back­ might not function like that of Mi. Importantly, whereas bone. Similar influences of acidic residues upstream of Mi is expressed in a few tissues other than pigment cells, the basic domain of E12 significantly suppress ho- the alternative splice form in melanocytes appears to be modimeric DNA binding in this b-HLH factor (Sun and unique (Hodgkinson et al. 1993) and may represent a Baltimore 1991), suggesting that comparable mecha­ truly melanocyte-specific b-HLH-ZIP factor. It will be nisms operate in other basic domain-containing tran­ important to examine MiT family expression in cells scription factors. The b-HLH-ZIP protein USF contains affected by mi mutations. Two of Mi's dimerization part­ a direct repeat peptide sequence that resembles an im­ ners have been shown to encode transcriptional inhibi­ munoglobulin hinge motif (Gregor et al. 1990). The pres­ tory activity. TFEC represses TFE3-dependent transcrip­ ence of proline near the amino terminus of all b-HLH- tion (Zhao et al. 1993) and an alternative splice form of ZIP basic domains suggests that the peptide backbone is TFE3 has also been shown to repress the longer tran­ kinked in such a fashion that the upstream amino acids scriptionally active form of TFE3 (Beckmann et al. 1990; may reach back in the vicinity of the basic domain. It is Roman et al. 1991). Thus, regulated MiT protein dimers also interesting that the 1212 mutation (Steingrimsson et might direct the tissue-specific expression of pigmenta­ al. 1994) occurs on the solvent exposed surface of the tion program genes. basic domain. Although this position is not likely to con­ tact DNA (Fisher et al. 1993; Ferre-D'Amare 1993; Ste­ Mi also functions in melanocytes as a lineage-re­ ingrimsson et al. 1994), it is strikingly conserved as a stricted survival factor. During melanocyte develoment, hydrophobic residue in all CACGTG-binding b-HLH- cells harboring mi mutations appear to die, rather than ZIP proteins and is usually an arginine in CAGCTG (e.g.) survive without producing pigment. The prospect binding ones (Dang et al. 1992). Because the b-HLH-ZIP that pigmentation enzymes and melanocyte survival basic domain is an intrinsically unstable a-helix (Fisher genes are downstream effectors of Mi represents one of et al. 1993; Ferre-D'Amare et al. 1994), interactions on very few known transcription factor targets for the this other face may affect DNA binding by influencing b-HLH-ZIP family. An understanding of the role of Mi a-helical folding. Although the mechanism by which the in melanocyte development may provide insight into upstream region influences DNA binding remains un­ pathways of cellular proliferation and death in which clear, it is likely to be functionally important because of other b-HLH-ZIP proteins, like Myc/Max, are known to its biological consequences in mice carrying the mf^ or play roles. MT"^ mutations. The mild "enhancing" phenotype of mf^ lacking the insert (Wolfe and Coleman 1964) and Materials and methods the interallelic complementation of Mf^^ (Griineberg 1952; Hollander 1968; Konyukhov and Osipov 1968; DNA clones Steingrimsson et al. 1994) might both be explained [mi^^ The wild-type mi cDNA derived from melan-c cells was ex- GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 12, 2021 - Published by Cold Spring Harbor Laboratory Press Hemesath et al. pressed in vitro from the clone pBS-Mi, which contains the ration from the initial (linear) slope of protein titrations under cDNA inserted into the EcoRl site of pBluescript SK -. This conditions of probe excess. Proteins were derived from in vitro cDNA lacks the 6-amino-acid alternative exon. Mutants corre­ translation reactions and were quantitated by determining sponding to the alleles mi (del 775-777), Mi°' (G776A), M^'' probe saturation in gel shift using probe of known specific ac­ (T764A), and the recessive alleles mi"^ (C916T), and mi"* tivity. Mi will aggregate with DNA in the absence of poly[d(I- C)]; therefore this nonspecific competitor was added to all re­ (G793A) (Steingrimsson et al. 1994) were generated by site-di­ rected mutagenesis of pBS-Mi using the method of Eckstein actions (as above). K^ measurements therefore reflect its pres­ according to the recommendations of the manufacturer (Amer- ence. Equilibrium conditions were established by incubation at sham). Templates for mi^"^, and constructs containing the 30°C for 75 min. Quantitation was carried out using a Phospho- 6-amino-acid alternative exon were expressed from PCR-de- rlmager (Molecular Dynamics). Immunoprecipitations were rived fragments made from wild-type as well as mi and Mi"^^ performed by mixing the various proteins under gel shift con­ mutant tissues. Expression templates were verified by DNA ditions (excluding poly [d(I-C)] and DNA probe) at 37°C for 1 hr, sequencing. TFEB was expressed from clone pTFEB-AA265 followed by addition of 3 [d of rabbit anti-TFEB antiserum and (Fisher et al. 1991). TFE3 in vitro expression vector was provided freshly washed protein A-Sepharose (Pharmacia), incubation at by Dr. T. Kadesch (Beckman et al. 1990). TFEC expression vec­ 4°C for 2 hr, and three washes with PBS containing 0.1% NP-40 prior to elution in loading buffer and SDS-PAGE. tor was provided by Dr. B. de Crombrugghe (Zhao et al. 1993). E47S was expressed from the plasmid pE47S (Murre et al. 1989). His fusion Mi was expressed from a plasmid containing the Transient transfections and luciferase assay BamHl-BamHl insert fragment from pBS-Mi inserted into the BamHl site of pET 15b (Novagen). For mammalian expression of NIH-3T3 cells were maintained in Dulbecco's modified Eagle Mi; the cDNA was cloned into the ffindlll and Xbal sites of medium supplemented with 5% calf serum/5% fetal calf se­ pRC-CMV (InVitrogen). The luciferase reporter plasmid was rum, 4 mM L-glutamine, 100 U/m l of penicillin, and 100 ixg/ml made by cloning an oligonucleotide containing four tandem re­ of streptomycin (GIBCO BRL). Cells were split 24—36 hr prior to peats of the M box (AGTCATGTGCT) into the Kpnl-Xhol sites transfection such that cells were ~60% confluent at the time of of the luciferase reporter plasmid pGL2 promoter (Promega). DNA addition, and were refed with fresh medium 8 hr prior to transfection. Transfections were carried out by calcium phos­ phate/DNA coprecipitation according to Kingston (1993) and Protein expression harvested after 24 hr. Three 6-cm plates were each transfected with 0.25 fjLg of luciferase reporter plasmid, 1 |xg of (3-galactosi- In vitro-translated proteins were made in rabbit reticulocyte dase control plasmid pRSV-p-Gal (Edlund et al. 1985), 4.7 jig of lysate (Promega) using RNA from in vitro transcription using cytomegalovirus (CMV)-driven expression vector pRC-CMV T7 RNA polymerase according to the manufacturer's recom­ (Invitrogen), and 4.05 |xg of carrier DNA pBS-SK (Stratagene). mendations (Pharmacia) for pBS-Mi and the corresponding mi, j^^wh^ j^^or^ j^^vit^ ^j ^-c e mutants as well as TFE3. Full-length At harvest, plates were washed once with phosphate-buffered Mi proteins were obtained by linearizing with Smal, and car- saline, lysed, and analyzed using a Monolight 2010 Luminom- boxy-terminal deletions at amino acids 319 and 261 were ob­ eter according to the recommendations of the manufacturer tained by linearizing with Xmnl and Avail, respectively. TFEB (Analytical Luminescence Laboratory, San Diego, CA). p-Galac- and E47S were transcribed using T3 RNA polymerase (Fisher et tosidase activity in cell lysates as a measure of relative trans­ al. 1991) (Pharmacia). Amino-terminal deletions and the DNA- fection efficiency was used to adjust luciferase data and was binding domain of TFEC were made by amplifying discrete frag­ assayed as described (Sambrook et al. 1989). ments using 5' primers that begin at the described residue and append an initiation ATG, Kozak sequence, and T3 RNA poly­ merase promoter (derived from the plasmid pBS-ATG, (Baldwin Acknowledgments et al. 1990) followed by transcription and translation in vitro. In We wish to thank Dr. Phillip Sharp for encouragement and sup­ vitro-translated proteins were quantitated by TCA precipitation port. Dr. Karen J. Moore for useful discussions, and Drs. T. and SDS-PAGE and equivalent quantities were added to gel shift Kadesch, B. deCrombrugghe and C. Miirre for plasmids. This assays. Recombinant TFEB was synthesized as described (Fisher work was supported in part by a grant from the Fimdacion In- et al. 1993). Recombinant His fusion Mi protein was synthe­ temacional Jose Carreras, and the National Cancer Institute sized in the bacterial strain BL-21, purified using nickel chelate under contract NOl-CO-74101 with ABL. chromatography (Qiagen), and eluted with 100 mM imidazole. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby Electiophoretic mobility shift assay, affinity measurements, marked "advertisement" in accordance with 18 USC section and immunoprecipitation 1734 solely to indicate this fact. DNA-binding assays were performed as described (Fisher et al. 1993) in 20-|xl reactions containing 5% glycerol, 100 mM KCl, References 10 mM Tris (pH 7.4), 1 mM DTT, and -5x10^^ cpm of ^^P-end- labeled probe DNA. In mixing experiments, separately trans­ Amati, B., M.W. Brooks, N. Levy, T.D. Littlewood, G.I. Evan, lated proteins were incubated at 37°C for 30 min prior to the and H. Land. 1993. Oncogenic activity of the c-Myc protein addition of probe DNA. CACGTG, CATGTG, and double point requires dimerization with Max. Cell 72: 233-245. mutant probes were used as described (Fisher et al. 1991). Poly- Anthony-Cahill, S.J., P.A. Benfield, R. Fairman, Z.R. Wasser- acrylamide gels (6% Tris-glycine-EDTA) were run and sub­ man, S.L. Brenner, W.F. Stafford, C. Altenbach, W.L. Hub- jected to autoradiography after drying. Competitors were pre­ bell, and W.F. DeGrado. 1992. Molecular characterization of pared as described previously (Fisher et al. 1991). Reactions helix-loop-helix peptides. Science 255: 979-983. probed with the CACGTG probe contained 1 jjig of poly[d(I-C)] Ayer, B. and R.N. Eisenman. 1993. A switch from Myc:Max to per 20-|xl reaction, whereas those containing CATGTG probe Mad:Max heterocomplexes accompanies monocyte/macro­ contained 0.5 |xg. K^ was determined by calculating half satu­ phage differentiation. 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Genes Dev. 1994, 8: Access the most recent version at doi:10.1101/gad.8.22.2770 This article cites 52 articles, 27 of which can be accessed free at: References http://genesdev.cshlp.org/content/8/22/2770.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Copyright © Cold Spring Harbor Laboratory Press

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