Identification of Aim-1 as the underwhiteMouse Mutant and Its Transcriptional Regulation by MITF

Animal pigmentation mutants have provided rich models for the identification of genes modulating pathways from melanocyte development to melanoma. One mouse model is theunderwhite locus, alleles of which manifest altered pigmentation of both eye and fur, sometimes in an age-dependent fashion. Here we show that the mouse homolog of a recently identified gene whose mutation produces Japanese gold-colored fish, medaka b, maps to the mouseunderwhite locus. We identify distinct mutations of this gene, known as Aim-1, in three underwhite mouse alleles and find that structure/function differences correlate with recessive versus dominant inheritance. The human ortholog of AIM-1 was originally identified as a melanocyte-restricted antigen that is recognized by autologous T cells from a patient with melanoma. We also provide evidence that AIM-1 is transcriptionally modulated by MITF, a melanocyte-specific transcription factor essential to pigmentation and a clinical diagnostic marker in human melanoma. Although AIM-1 appears to reside downstream of MITF, chromatin immunoprecipitations do not reveal binding of MITF to a 5′-flanking region containing histone 3 acetylation, indicating that MITF either acts indirectly on AIM-1 or it binds to a remote regulatory sequence. Nevertheless, MITF links AIM-1 expression and theunderwhite phenotype to a transcriptional network central to pigmentation in mammals. Animal pigmentation mutants have provided rich models for the identification of genes modulating pathways from melanocyte development to melanoma. One mouse model is theunderwhite locus, alleles of which manifest altered pigmentation of both eye and fur, sometimes in an age-dependent fashion. Here we show that the mouse homolog of a recently identified gene whose mutation produces Japanese gold-colored fish, medaka b, maps to the mouseunderwhite locus. We identify distinct mutations of this gene, known as Aim-1, in three underwhite mouse alleles and find that structure/function differences correlate with recessive versus dominant inheritance. The human ortholog of AIM-1 was originally identified as a melanocyte-restricted antigen that is recognized by autologous T cells from a patient with melanoma. We also provide evidence that AIM-1 is transcriptionally modulated by MITF, a melanocyte-specific transcription factor essential to pigmentation and a clinical diagnostic marker in human melanoma. Although AIM-1 appears to reside downstream of MITF, chromatin immunoprecipitations do not reveal binding of MITF to a 5′-flanking region containing histone 3 acetylation, indicating that MITF either acts indirectly on AIM-1 or it binds to a remote regulatory sequence. Nevertheless, MITF links AIM-1 expression and theunderwhite phenotype to a transcriptional network central to pigmentation in mammals. chromatin immunoprecipitation assay rapid amplification of cDNA ends A recent study reported a novel transporter protein AIM-1 that is responsible for the pigment phenotype of medaka b gold-fish mutants (1Fukamachi S. Shimada A. Shima A. Nat. Genet. 2001; 28: 381-385Crossref PubMed Scopus (189) Google Scholar). In this fish, mutations in Aim-1 produce albino-like depigmentation of black melanophores, leaving gold-red xanthophores, white leukophores, and silver iridophores as the remaining pigmented cells. The gene product is predicted to be a 12-transmembrane-transporter protein that shows homology to yeast and plant sucrose transporters (1Fukamachi S. Shimada A. Shima A. Nat. Genet. 2001; 28: 381-385Crossref PubMed Scopus (189) Google Scholar). Human AIM-1 has been previously identified as a melanoma antigen that was recognized by autologous, patient-derived T cells (2Harada M. Li Y.F. El-Gamil M. Rosenberg S.A. Robbins P.F. Cancer Res. 2001; 61: 1089-1094PubMed Google Scholar). Its expression was shown to be restricted to the melanocyte lineage by Northern analysis (2Harada M. Li Y.F. El-Gamil M. Rosenberg S.A. Robbins P.F. Cancer Res. 2001; 61: 1089-1094PubMed Google Scholar). MurineAim-1 is located on chromosome 15 in a region containing the pigmentation locus, underwhite. A series of mouse mutants has been described for this locus. All are characterized by various degrees of pigment reduction (from partial to complete albinism) in the eyes and fur (3Sweet H.O. Brilliant M.H. Cook S.A. Johnson K.R. Davisson M.T. J. Hered. 1998; 89: 546-551Crossref PubMed Scopus (32) Google Scholar). MITF is a tissue-restricted transcription factor essential to melanocyte development. It binds the canonical E-box sequence CACGTG as well as the non-palindromic sequence CACATG. The major pigmentation hormone, melanocyte-stimulating hormone, up-regulates MITF expression through cAMP signaling followed by cAMP-response element-binding protein phosphorylation and activation of the melanocyte-specific MITF promoter and may modulate multiple pigmentation genes through up-regulating MITF expression (4Price E.R. Horstmann M.A. Wells A.G. Weilbaecher K.N. Takemoto C.M. Landis M.W. Fisher D.E. J. Biol. Chem. 1998; 273: 33042-33047Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 5Bertolotto C. Abbe P. Hemesath T.J. Bille K. Fisher D.E. Ortonne J.P. Ballotti R. J. Cell Biol. 1998; 142: 827-835Crossref PubMed Scopus (422) Google Scholar). The three major pigmentation enzymes tyrosinase, TYRP1, and DCT, all contain consensus MITF DNA binding elements that are conserved across species and are thought to be transcriptional targets of MITF (6Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (578) Google Scholar, 7Bentley N.J. Eisen T. Goding C.R. Mol. Cell. Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (431) Google Scholar, 8Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Mol. Cell. Biol. 1994; 14: 8058-8070Crossref PubMed Scopus (368) Google Scholar). In humans, germline heterozygous MITF mutation produces the pigmentation-deafness condition Waardenburg Syndrome IIA (9Tassabehji M. Newton V.E. Read A.P. Nat. Genet. 1994; 8: 251-255Crossref PubMed Scopus (580) Google Scholar) and Tietz syndrome (10Amiel J. Watkin P.M. Tassabehji M. Read A.P. Winter R.M. Clin. Dysmorphol. 1998; 7: 17-20Crossref PubMed Scopus (87) Google Scholar, 11Smith S.D. Kelley P.M. Kenyon J.B. Hoover D. J. Med. Genet. 2000; 37: 446-448Crossref PubMed Scopus (123) Google Scholar), manifesting pigmentation disturbances and deafness due to inner ear melanocyte deficiency (12Price E.R. Fisher D.E. Neuron. 2001; 30: 15-18Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Interestingly, MITF expression is usually (if not always) maintained in human melanoma specimens, and it is increasingly used as a histopathologic marker for melanoma diagnosis (13Busam K.J. Iversen K. Coplan K.C. Jungbluth A.A. Am. J. Surg. Pathol. 2001; 25: 197-204Crossref PubMed Scopus (142) Google Scholar, 14King R. Weilbaecher K.N. McGill G. Cooley E. Mihm M. Fisher D.E. Am. J. Pathol. 1999; 155: 731-738Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Koch M.B. Shih I.M. Weiss S.W. Folpe A.L. Am. J. Surg. Pathol. 2001; 25: 58-64Crossref PubMed Scopus (80) Google Scholar, 16Miettinen M. Fernandez M. Franssila K. Gatalica Z. Lasota J. Sarlomo-Rikala M. Am. J. Surg. Pathol. 2001; 25: 205-211Crossref PubMed Scopus (154) Google Scholar, 17Salti G.I. Manougian T. Farolan M. Shilkaitis A. Majumdar D. Das Gupta T.K. Cancer Res. 2000; 60: 5012-5016PubMed Google Scholar). We BLASTed the human AIM-1 mRNA sequence (accession numberAF172849) against the human genome and located the gene to chromosome 5. Examination of the homologous region in the mouse genome revealed a previously described hypopigmentation locus, underwhite. Here experiments were carried out to explore a potential link betweenAim-1 and the underwhite mutants. Distinct mutations in Aim-1 were found in three underwhitealleles, including a frameshift, as compared with wild-type controls. In addition, evidence is presented that suggests that the global transcriptional regulator of pigmentation, MITF, also resides upstream of Aim-1, linking this gene to the major pigmentation pathway in melanocytes. Human AIM-1 mRNA sequence (accession number AF172849) was BLASTed against the Human Genome. MouseAim-1 mRNA sequence (accession number AF360357) was BLASTed against the mouse Trace Archive. Genomic DNA from C57BL/6J and homozygous uw, uwd, andUwDbr mice was obtained from the Jackson Laboratory (Bar Harbor, ME). Genomic DNA from adult male BALB/c kidney tissue was purchased from CLONTECH. Primer pairs spanning each exon were designed as follows. Exon 1: 5′-CTG AGG ACC ACG CAA GAA GGC TAT T-3′ and 5′-CCA GGC TCG GGG TCA TCC AAA GGT G-3′. Exon 2: 5′-TAA AAC CCA ACC TAC AAA ACC AAA ACA-3′ and 5′-GGC ACT TCC TAT CAA CTG ACC CAT TC-3′. Exon 3: 5′-GAA GGT CTG TGC ATG GTG GGA AAT AAA C-3′ and 5′-AGG CAA GAG AAC CAC TGA GGC ACA AAA T-3′. Exon 4: 5′-TCT GGC TGT GGC TCT GAC TCT GA-3′ and 5′-CAT GCC ATT CCT GTT TCC ACT TAG-3′. Exon 5: 5′-GTG CTG TCT GCT TGA ACT CTG G-3′ and 5′-ATA TAA AAT CTG GAT CCT GCT GCT A-3′. Exon 6: 5′-CTC AGT ATC AAA GGA AGT CGT CTA AAA-3′ and 5′-TTG GGG TCA CTA TCA TTG TCC TAA AA-3′. Exon 7: 5′-GCC CTG TGC GCT AGT GCC CTG TA-3′ and 5′-AGT TGC TGT GCT TTC GGA ATG AGA CCT-3′. Chromatin immunoprecipitation assay (ChIPs)1 was performed in human primary melanocytes (provided by Dr. Ruth Halaban, Yale University), SKMEL5, or IMR90 cells (ATCC) grown in logarithmic phase. Cells were harvested by scraping, homogenized in a hypotonic buffer (10 mm Tris-HCl, pH 7.4, 15 mm NaCl, 60 mm KCl, 1 mm EDTA, 0.1% Nonidet P40, 5% sucrose, 1× CompleteTM proteinase inhibitor mixture (Roche)) on ice using a Dounce homogenizer. The nuclei were isolated by centrifugation onto a 10% sucrose pad and then cross-linked with 1% formaldehyde in phosphate-buffered saline for 20 min at room temperature with gentle shaking. Nuclei were then spun down and resuspended in ChIPs buffer (10 mm Tris-HCl pH 7.4, 100 mm NaCl, 60 mm KCl, 0.1% Nonidet P40, 1× proteinase inhibitors) and sonicated by two 1-min pulses using a Fisher dismembranator fitted with a micro-tip on ice. Antibodies against USF1 (C-20, Santa Cruz), USF2 (N-18, Santa Cruz), c-Myc (N-262X, Santa Cruz), polyclonal rabbit anti-MITF, or acetylated Histone H3 (Upstate) were then added to a 10-fold ChIPs buffer diluted sample and incubated on a nutator for 3 h at room temperature. Subsequently, Ultralink protein A/G beads (Pierce) were added to the sample and a control sample and incubated for an additional hour. Immunoprecipitates were then washed twice with ChIPs buffer, twice with 500 mmNaCl ChIPs buffer and once with TE, pH 8. The immunoprecipitates were released from the beads by incubating at 65 °C for 20 min in 1% SDS/TE, and proteins were digested by proteinase K treatment side-by-side with an additional unprecipitated sample as input control. Cross-links were released by heating at 70 °C for 10 h, DNA recovered by extraction with phenol and chloroform at high salt (0.6 m sodium acetate, pH 8), and then ethanol precipitated. Semi-quantitative PCR was then performed on samples to amplify fragments spanning the 5′- or 3′-adjacent region to the E box repeat (see Fig. 2a). The forward and reverse primers for the 5′-region are 5′-TGT TAA GTA CCA CGA GGA GAA ATA-3′ and 5′-ACA TGG CCG TGA GGT AAT AAA G-3′, respectively, (annealing temperature 59 °C). The primers for the 3′-region are 5′-CAG CAC CAC CCC CTC CCT CTC ATC ATA AC-3′ and 5′-CCA CAG AGT CAA AGG GGC CAT CAT CAG C-3′ (annealing temperature 65 °C). The primers for the MITF-positive ChIP control region (intron 1 of PMEL17) are 5′-CAT AAG ATA CCC CAT TCT TTC TCC ACT T-3′ and 5′-GAG AAT GTG GTA TTG GGT AAG AAC AC-3′ (annealing temperature 57 °C). PCR was carried out for 45 s at 94 °C, 45 s at annealing temperatures as indicated for each primer set, and 90 s at 72 °C for a total 35 cycles with Taq polymerase (Fisher). Null adenovirus was purchased from Q-BIOgene. Adenoviruses encoding wild-type MITF, dominant-negative mutant MITF, or vector control encoding a fusion of green fluorescence protein-Wee1 for nuclear localization, were generated as previously described (18Wu M. Hemesath T.J. Takemoto C.M. Horstmann M.A. Wells A.G. Price E.R. Fisher D.Z. Fisher D.E. Genes Dev. 2000; 14: 301-312PubMed Google Scholar). Briefly, 106 human SKMEL5 melanoma cells were plated per 100-mm plate. On the second day, cells were overlaid with 2 ml of serum-free F10 media containing 10 mm MgCl2, and concentrated adenovirus was added at multiplicity of infection of 100 for each virus. The cells were incubated at 37 °C for half an hour after which virus was removed and fresh full media was added. Total RNA was isolated with RNAqueousTM-4PCR kit (Ambion 1914) at 48 and 72 h after infection. The real-time PCR primers for human AIM-1 were 5′-CCTGGGCTTTCTGGTCAACA-3′ and 5′-ACCGCAGACGCTGTGATCA-3′. The probe for human AIM-1 were 5′-6-FAM-AGCCGGGACCGTTGTCGTCG-TAMRA-3′ (PE Biosystems). The total volume of each reaction is 25 μl including 12.5 μl 2× Master Mix without UNG (uracil-N-glycosylase), 0.625 μl MultiScribe Reverse Transcriptase and RNase inhibitor (PE Biosystems), 0.5μl of each primer (10 μm stock), 0.25 μl of the probe (5 μm stock) and 1 μl of the template at 100 ng/μl. Reverse transcription proceeded at 48 °C for 30 min. Then 40 cycles of PCR reaction were carried out at 95 °C for 15 s and at 60 °C for 1 min. Real-time PCR was carried out using ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with analysis using the integrated Sequence Detection System Software Version 1.7. We used human map viewer (ncbi.nlm.nih.gov/) to determine that the human ortholog of Aim-1 is located on chromosome 5p. We identified a region homologous to the human Aim-1 locus on mouse chromosome 15 and noted that this site maps to the murineunderwhite locus (ncbi.nlm.nih.gov/Homology/and Ref. 19Blake J.A. Eppig J.T. Richardson J.E. Bult C.J. Kadin J.A. Nucleic Acids Res. 2001; 29: 91-94Crossref PubMed Scopus (59) Google Scholar). The complete mouse Aim-1 cDNA sequence (accession number AF360357) was BLASTed against the mouse Trace Archive to identify the exon/intron boundaries (TableI). Primer pairs located in the intron regions were designed to span individual exons. Each exon was amplified from the genomic DNA of uw, uwd andUwDbr mice as well as the control C57BL/6J and BALB/c strains. All PCR products matched the predicted lengths (data not shown). DNA sequencing identified mutations in all three mutant alleles, but none of their wild-type controls (Fig.1a). The uw mutant was found to harbor a 7-base pair deletion in Exon 3 ofAim-1 (Fig. 1a), which results in a 43-amino followed by a at (Fig. and The uw a protein that the of the predicted The uwd mutant a mutation from T to in Exon of 1a), which to a mutation from a conserved to in the (Fig. b The dominant a from to A in the second exon which a mutation of from medaka to in the (Fig. b and identify Aim-1 as the mutant gene in of human and mouse Aim-1 exon sequence of sequence of in a of box DNA elements MITF is thought to modulate expression of major pigmentation genes including tyrosinase, TYRP1, and (6Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (578) Google Scholar, 7Bentley N.J. Eisen T. Goding C.R. Mol. Cell. Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (431) Google Scholar, 8Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Mol. Cell. Biol. 1994; 14: 8058-8070Crossref PubMed Scopus (368) Google Scholar). experiments were to MITF AIM-1 expression in the melanocyte MITF was in human melanoma cells by with a series of (see including a control green fluorescence protein fusion from the wild-type MITF, or dominant-negative MITF which but DNA binding by (6Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (578) Google Scholar, M. Hemesath T.J. Takemoto C.M. Horstmann M.A. Wells A.G. Price E.R. Fisher D.Z. Fisher D.E. Genes Dev. 2000; 14: 301-312PubMed Google Scholar). revealed that expression of MITF proteins is at 48 h after infection and at h (data not shown). assay that the DNA binding of the wild-type protein and the dominant-negative of the mutant protein are this of (data not shown). the of MITF on AIM-1 RNA was harvested from cells 48 and 72 h after infection. Real-time PCR was performed on the RNA and AIM-1 expression were to PCR shown in Fig. wild-type MITF AIM-1 expression control dominant-negative MITF results were also in human primary melanocytes (data not shown). were at multiple The that MITF modulate the expression of AIM-1 in human melanoma cells. Examination of the upstream region of the human Aim-1 gene revealed a sequence containing E or repeat in this It is this promoter is conserved in the A J. P. P. Chem. PubMed Scopus Google Scholar) on this repeat against J. Biol. 1998; 8: PubMed Scopus Google Scholar, J. Genet. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Genet. Dev. 1999; PubMed Scopus Google Scholar) homology to known human a against the human genome revealed a of this repeat in genome by contain or One for rapid of or a is repeat It appears for that the repeat be from a repeat by MITF and E box binding as and USF2 on the AIM-1 5′-flanking chromatin immunoprecipitation was carried out on human primary human melanoma cells and IMR90 cells were designed that span the 5′- or 3′-region of the repeat In primary but not PCR products were amplified in samples with an against acetylated histone a of transcriptionally In addition, PCR were performed on total RNA from all three AIM-1 were in primary human SKMEL5 melanoma cells but not IMR90 (Fig. results are with melanocyte-restricted expression of AIM-1 (2Harada M. Li Y.F. El-Gamil M. Rosenberg S.A. Robbins P.F. Cancer Res. 2001; 61: 1089-1094PubMed Google Scholar). the various proteins including MITF, do not with this region the consensus control ChIPs for MITF are shown for a genomic (Fig. results were also obtained from SKMEL5 melanoma of acetylated histone H3 not as in the differences in transcriptional of AIM-1 in this melanoma results that MITF or proteins do not the E-box repeat in the melanocyte from this region to and to in melanocytes but to MITF (data not shown). that MITF not modulate AIM-1 transcription at the but a remote binding or through a transcriptional It is also that the altered RNA of AIM-1 by MITF in RNA an has not previously been described for The described identify the Aim-1 gene as the locus responsible for the mouse underwhite of of the protein in the mutant to complete of pigmentation and the pigment deficiency of the three alleles In the mutation produces a in the uw mutation is a which as a the uwd mutation a partial of that not with the product of the remaining wild-type In addition, that not for this gene, a with pigment as J.A. Eppig J.T. Richardson J.E. Bult C.J. Kadin J.A. Nucleic Acids Res. 2001; 29: 91-94Crossref PubMed Scopus (59) Google Scholar), of pigmentation reveal differences that are not The dominant of suggests that Aim-1 in a with as a or remote sequence homology to the sucrose transporter (1Fukamachi S. Shimada A. Shima A. Nat. Genet. 2001; 28: 381-385Crossref PubMed Scopus (189) Google Scholar) whose are not well or has been reported for transporter and transporters in yeast D. Cell Biol. 2001; PubMed Scopus Google Scholar). mutation a it is that the altered of the a salt or that to in a that not dominant this mutation produces a phenotype in as compared with the indicating that in homozygous the mutant protein wild-type The transcriptional described in this study is on using (1Fukamachi S. Shimada A. Shima A. Nat. Genet. 2001; 28: 381-385Crossref PubMed Scopus (189) Google Scholar). as primer be to the transcriptional site in mammals. is of the medaka AIM-1 mRNA an region that is not in the human and mouse is to the ATG for human or that the sequence may not contain the of a E-box containing the human 5′-flanking region not correlate with binding by the protein MITF, the that MITF AIM-1 expression MITF AIM-1 expression an as an transcription or an located at a from this It is also that the reported transcriptional site is in which a promoter region may be to MITF binding and downstream of MITF it the of multiple genes of known in the pigmentation of which are as melanoma identification of AIM-1 as a pigmentation gene suggests the that human mutations in the factor The uw age-dependent in the eye pigmentation phenotype and complete of pigment in the fur M. 30: Scholar, A.L. K. S. Brilliant M.H. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, gene or eye pigment at and development of eye pigment the of J. Clin. Genet. 29: PubMed Scopus Google Scholar). In addition, this was M. Brilliant and the identification of two of the underwhite alleles described in this study and reported the of theunderwhite locus to human Davisson M.T. Brilliant M.H. Am. J. Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). underwhite mice to provide a model for this or of in We Dr. for on We also Dr. Ruth for human primary melanocyte is for to their of human and

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