Gene 552 (2014) 165–175

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Alternative splicing of the sheep MITF gene: Novel transcripts detectable in skin Siva Arumugam Saravanaperumal ⁎, Dario Pediconi, Carlo Renieri, Antonietta La Terza ⁎⁎ Animal and Molecular Ecology Lab, School of Biosciences and Veterinary Medicine, University of Camerino, via Gentile III da Varano, Camerino, Macerata 62032, Italy

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Article history: Received 1 January 2014 Received in revised form 12 September 2014 Accepted 15 September 2014 Available online 18 September 2014 Keywords: Alternative splicing (AS) Exon skipping Merino sheep Microphthalmia-associated transcription factor (MITF) Splice variant

a b s t r a c t Microphthalmia-associated transcription factor (MITF) is a basic helix–loop–helix leucine zipper (bHLH-LZ) transcription factor, which regulates the differentiation and development of melanocytes and pigment cell-specific transcription of the melanogenesis enzyme genes. Though multiple splice variants of MITF have been reported in humans, mice and other vertebrate species, in merino sheep (Ovis aries), MITF gene splicing has not yet been investigated until now. To investigate the sheep MITF isoforms, the full length mRNA/cDNAs from the skin of merino sheep were cloned, sequenced and characterized. Reverse transcriptase (RT)-PCR analysis and molecular prediction revealed two basic splice variants with (+) and without (−) an 18 bp insertion viz. CGTGTATTTTCCCCACAG, in the coding region (CDS) for the amino acids ‘ACIFPT’. It was further confirmed by the complete nucleotide sequencing of splice junction covering intron-6 (2463 bp), wherein an 18 bp intronic sequence is retained into the CDS of MITF (+) isoform. Further, full-length cDNA libraries were enriched by the method of 5′ and 3′ rapid amplification of cDNA ends (RACE-PCR). A total of seven sheep MITF splice variants, with distinct N-terminus sequences such as MITF-A, B, E, H, and M, the counterparts of human and mouse MITF, were identified by 5′ RACE. The other two 5′ RACE products were found to be novel splice variants of MITF and represented as ‘MITF truncated form (Trn)-1, 2’. These alternative splice (AS) variants were illustrated using comparative genome analysis. By means of 3′ RACE three different MITF 3′ UTRs (625, 1083, 3167 bp) were identified and characterized. We also demonstrated that the MITF gene expression determined at transcript level is mediated via an intron-6 splicing event. Here we summarize for the first time, the expression of seven MITF splice variants with three distinct 3′ UTRs in the skin of merino sheep. Our data refine the structure of the MITF gene in sheep beyond what was previously known in humans, mice, dogs and other mammals. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Many pigmentation mutants are phenotypically (N800 alleles) profound, but remain mechanistically uncharacterized (Bennett and Lamoreux, 2003). In sheep, the candidate genes for recessive black (ASIP) (Parsons et al., 1999a, 1999b), dominant black (MC1R) (Vage Abbreviations: MITF, microphthalmia-associated transcription factor; c-KIT, Mast/stem cell growth factor receptor (SCFR) or proto-oncogene c-Kit or tyrosine-protein kinase Kit or CD117; SCF, stem cell factor; bHLH-LZ, basic helix–loop–helix leucine zipper; ASIP, Agouti signaling protein; MC1R, melanocortin 1 receptor; TYRP1, tyrosinase-related protein 1; Tyr, tyrosinase; Dct/Tyrp-2, dopachrome-tautomerase/tyrosinase-related protein 2; RACE-PCR, rapid amplification of cDNA ends-polymerase chain reaction; RT, Reverse Transcription; dNTP, deoxyribonucleoside triphosphate; DEPC, diethyl pyrocarbonate; DMSO, dimethyl sulfoxide; TdT, terminal deoxynucleotidyl transferase; dCTP, deoxycytidine triphosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; rRNA, ribosomal RNA; RNA, ribonucleic acid; miRNA/miRs, microRNAs; gDNA, genomic DNA. ⁎ Correspondence to: S.A. Saravanaperumal, Department of Physiology and Biomedical Engineering Enteric NeuroScience Program, Mayo Clinic, 200 1st Street SW, Guggenheim 8-98, Rochester, MN 55905, USA. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (S.A. Saravanaperumal), [email protected] (D. Pediconi), [email protected] (C. Renieri), [email protected] (A. La Terza).

http://dx.doi.org/10.1016/j.gene.2014.09.031 0378-1119/© 2014 Elsevier B.V. All rights reserved.

et al., 1999, 2003) and Brown (TYRP1) (Beraldi et al., 2006) have been found; these genes are known to influence pigmentation or pigment synthesis level. Many white spotting traits have been identified in mouse and man; and 10 of the responsible genes have been cloned (Baxter et al., 2004). It has been hypothesized that the gene for white phenotype in merino sheep is one of these loci (Renieri et al., 2008). Through mutations in microphthalmia-associated transcription factor (MITF, microphthalmia) (Tachibana, 2000), c-KIT (Dominant White Spotting) and stem cell factor (SCF, Steel), it is possible to obtain completely white live animals (Bennett and Lamoreux, 2003; Baxter et al., 2004; Hoekstra, 2006). In merino sheep, a novel splice variant of SCF gene involving premature termination of transcription has been reported (Saravanaperumal et al., 2012). Microphthalmia-associated transcription factor (MITF) and its human counterpart huMITF (Tachibana et al., 1994) contain a basic helix–loop–helix-leucine zipper (bHLH-LZ) structure (Hodgkinson et al., 1993; Hughes et al., 1993) required for DNA binding and dimerization (Hallsson et al., 2000; Udono et al., 2000). MITF plays important roles in the development and differentiation of neural crest-derived melanocytes (Goding, 2000; Lekmine et al., 2007), bone marrow-derived mast cells (Hodgkinson et al., 1993; Shahlaee et al., 2007), osteoclasts

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(Mansky et al., 2002) and optic cup-derived retinal pigment epithelium (RPE) (Bharti et al., 2008). Mitf is encoded by the Mitf (mi) locus in mice (Hodgkinson et al., 1993) and, when mutated, shows peculiar phenotypes, i.e., white coat color and microphthalmia (Tachibana et al., 1992), and leads to defects in melanocytes, the retinal pigmented epithelium (RPE), mast cells and osteoclasts (Hodgkinson et al., 1993; Widlund and Fisher, 2003; Steingrímsson et al., 2004). In humans, germline heterozygous mutations of the MITF gene are associated with the congenital pigmentation/deafness condition, Waardenburg Syndrome (WS) type 2A (OMIM #193510) (Hughes et al., 1994; Tassabehji et al., 1994; Nobukuni et al., 1996), in which affected individuals display variable degrees of pigmentation, and associated deafness due to melanocyte defects in the inner ear (Price and Fisher, 2001). A subtype of WS type 2A, namely Tietz syndrome (OMIM #103500), is characterized by a more severe phenotype wherein affected patients display more severe deafness and pigment disturbances (Amiel et al., 1998; Smith et al., 2000). Consistent with its central role in melanocyte development (Steingrímsson et al., 2004), and as a regulator of gene expression, Mitf is primarily located in the nucleus (Takebayashi et al., 1996), where it can activate and regulate expression of three pigmentation enzyme genes: tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1) and dopachrome-tautomerase (Dct/Tyrp-2) (Bentley et al., 1994; Yasumoto et al., 1994; Yasumoto and Shibahara, 1997). Alternatively spliced transcript variants of human MITF encoding different isoforms have been identified. In humans, MITF consists of at least eight isoforms, referred to as MITF-M, MITF-H, MITF-A, MITF-B, MITF-C, MITF-D, MITF-E and MITF-J (Source: NCBI, Ensembl). Human, mouse and dog MITF genes contain multiple promoters and first exons, generating multiple MITF isoforms, suggesting the functional diversity of these isoforms in a variety of tissues. Each isoform of MITF is differentiated by its unique N-terminus encoded by separate first exons designated as 1M, 1H, 1A, 1B, 1C, 1D, 1E and 1J. All the isoforms of MITF share the

entire downstream region containing a transactivation domain and a bHLH-LZ domain encoded by exons 2–9 (Shibahara et al., 2001; Hallsson et al., 2007; Tsuchida et al., 2009). Isolation of MITF gene and knowledge of their structure will allow for further studies into the regulation of gene expression in the sheep skin biology. Although many isoforms have been described for MITF genes in humans, mice, and dogs, there is not currently experimental evidence in sheep for the MITF transcript profile, especially in the skin. Very recently an updated version of this gene became available for sheep in NCBI (Gene ID: 101115163, PREDICTED version Oar_v3.1 updated on 03-June-2014). There exist two partial uncharacterized entries one each for mRNA (EU028312.1) and DNA (GAAI01000006.1, from the heart). Sheep nucleotide BLAST against eight of huMITF variants (GenBank) resulted in 7 non-normalized expressed sequence tags (ESTs), from parotid lymph nodes, wool follicle, healing fracture callus and endometrium. To our knowledge this is the first experimental evidence of the complete MITF transcript profile detectable in the skin from merino sheep. In an effort, to better characterize the mRNA/cDNA structure of MITF in the skin of merino sheep we performed cDNA and gDNA cloning, sequencing, and gene expression by RT-PCR analysis. In this study, we isolated and characterized seven MITF splice variants from the skin that code for distinct N-terminus sequences (Fig. 1). We isolated two novel splice variants of MITF, designated as ‘MITF truncated form (Trn)-1, 2’. The two basic splice variants of MITF transcript variants, ‘(+) and (−)’, the commonly known homologs of MITF in other mammals, are also presented here. We also demonstrated that the relative expression of MITF ‘(+) and (−)’ mRNA in the skin is mediated by an intron-6 splicing event. In addition, three different sizes of MITF 3′ UTRs were identified and characterized. This manuscript discusses the complete structural variation of MITF mRNA (Fig. 7), and putative AS events on the intron-1/2 of the MITF gene by comparative genomics. This work provides a basis for understanding MITF functions in the skin of sheep.

Fig. 1. Schematic representation of the seven MITF splice variants detected in the skin of sheep. Translated sheep MITF cDNA sequences aligned with reference to human MITF revealed 7 alternative splice variants. In the above picture, the alternative splicing (AS) mechanism of the sheep MITF gene is depicted with the possible splice donor and acceptor site (follow the key to symbols). BLASTN and BLASTP analyses revealed that seven different N-termini of sheep MITF cDNA, identified by 5′ RACE (see Fig. 5A) are shown with corresponding AS notation. Also, 3 out of 4 validated alternative poly-adenylation sites (see the notation) were also identified by 3′ RACE (see Fig. 5B).

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2. Materials and methods 2.1. Collection of skin biopsies and blood Skin biopsies were collected from uncolored (white) and colored (black and brown) individual merino sheep using biopsy punches (8 mm diameter), treated and stored in RNAlater (Sigma-Aldrich), and immediately frozen in liquid nitrogen until RNA extraction. Blood samples were collected from the jugular vein of the same individuals with PAXgene Blood DNA Tubes (PreAnalytix/Qiagen) via a standard phlebotomy technique, processed immediately in the lab according to the manufacturer's protocol for the DNA isolation; DNA aliquots were stored at − 80 °C. Samples were collected and recorded according to the farm technicians from the following farms: “La Campana” located in the proximity of Montefiore dell'Aso (Ascoli Piceno, Marche) and “La Meridiana” in Umbertide (Perugia, Umbria), Italy.

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The primer pairs specific to MITF isoform (+/−), used for amplification of the open reading frame (ORF) corresponding to the ~986 bp of the sheep MITF cDNA, were designed based on the coding sequence homology among cows (GenBank Acc. No. NM_001001150.1), pigs (NM_ 001038001.1), dogs (NM_001003337.1), horses (NM_001163874.1), humans (NM_000248.3), chimpanzees (XM_001138431.1), mice (NM_008601.3) and rats (NM_001191089.1) using Primer3 software (Rozen and Skaletsky, 2000). The remaining 5′ and 3′ RACE MITF gene specific primer pairs were deduced from the 986 bp cDNA coding sequence (CDS) fragment in order obtain the full-length cDNAs. All the designed primer pairs were checked with the online software tools (http://www.sigmaaldrich.com/configurator/servlet/DesignTool? prod_type=STANDARD; Kalendar, 2010) before making an order with Sigma. The primers used in this study were synthesized and purchased from Sigma-Aldrich, Italy. 2.5. Rapid amplification of cDNA ends (5′ and 3′ RACEs)

2.2. Ethics statement In agreement with the new European Directive on the protection of animals used for scientific purposes (Directive 2010/63/EU, Article 15, Annex VIII), all animal procedures used in the study are classified as ‘mild’ (i.e. procedures with no significant impairment of the wellbeing or general condition of the animals) and were preemptively approved by the Animal Ethics Committee of the University of Camerino. 2.3. RNA and DNA isolation and quantification Total RNAs were extracted from the stored skin biopsies using TRI Reagent (Sigma-Aldrich, Italy) according to the manufacturer's instructions and treated with RNase-free DNase (Fermentas, Italy) to remove contaminated DNAs. Genomic DNAs were isolated from the blood samples with the PAXgene Blood DNA kit (PreAnalytix/Qiagen) following the given handbook protocol. Qualitative assessments of the purified DNAs and RNAs were determined utilizing the Genesys 10 UV Spectrophotometer (Thermo Electron Corporation, Madison, USA). 2.4. cDNA synthesis and RT-PCR amplification cDNAs were synthesized from total RNA extracted from the skin of the merino sheep. Reverse Transcription (RT) from 1–1.5 μg of RNA in a total volume of 20 μl containing 50 pmol oligo(dT) (18-mer) or oligo(dT)18 modified primer, 0.5 mM deoxyribonucleoside triphosphate (dNTPs), 1 × RT buffer, 20 U of RNase inhibitor and 200 U PrimScript™ Reverse Transcriptase (Takara Biotech, Japan) or StrataScript™ Reverse Transcriptase (Agilent, Stratagene, Italy) according to the manufacturer's instructions. The reaction was incubated for 60 min at 42 °C and then heated at 70 °C for 15 min, and cooled on ice. Subsequently, 0.5–0.7 μl of the first strand cDNA reaction was used for PCR amplification. Reactions were performed in 25 μl volume containing 1 × PCR bufffer, 1.5 mM MgCl2, 2.0 mM dNTPs, 0.3–0.5 μM gene specific primers (see Table S2), 20–30 ng/μl cDNA and 1.5 U of proofreading Easy-A HighFidelity PCR Cloning Enzyme (Agilent, Stratagene, Italy). Three-step RTPCR amplification was performed in a MyCycler™ Thermal Cycler (BioRad Laboratories Inc., Hercules, California, USA), with the following program: initial denaturation at 95 °C for 3 min, 5 primary cycles of 94 °C for 1 min, followed by an annealing temperature (Ta°C) 3–5 °C below the melting temperature (Tm) of the gene specific primer, 72 °C for 1 min. This was followed by 25 consecutive cycles of 94 °C for 15–30 s, annealing temperature (Ta°C) for 15–30 s, and 72 °C for 20–30 s with a final extension at 72 °C for 10 min and a hold temperature at 4 °C. PCR cycling conditions, especially Ta and timing interval (viz. 45 s to 1 min for amplicon sizes over 500–1000 bp) vary across primer sets and the expected size of amplicons (see Table S2 for details).

5′ and 3′ RACE experiments to isolate and determine the sheep fulllength MITF cDNA(s) were performed following the instructions of 5′ (v. 2.0) and 3′ (v. E) RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Italy). 5′ RACE cDNAs were reverse transcribed from 1–1.5 μg of RNA in a total volume of 20 μl containing 2–2.5 pmol MITF gene specific splice variant primer(s) (Table S2), 0.5 mM dNTPs, 1 × RT buffer, 20 U of RNase inhibitor, 200 U of PrimScript™ Reverse Transcriptase (Takara Biotech, Japan) and StrataScript™ Reverse Transcriptase (Agilent, Stratagene, Italy) according to the manufacturer's instructions. The reaction was incubated for 60 min at 50 °C and then heated at 70 °C for 15 min, cooled on ice and stored at −20 °C. The primer combinations used to amplify different MITF splice variants (5′/N-terminus) are provided in Table S2. Forward adapter primer sequences were retrieved from the 5′ RACE kit, Invitrogen, USA and synthesized by Sigma-Aldrich, Italy. The PCR amplification was carried out as described above for 36 cycles and the cycling conditions (e.g., Ta and timing interval) varied with 5′ RACE primer sets; see Table S2 for details about the expected size of amplicons. Occasionally, 0.67 M homoectoine was used for GC-rich 5′ RACE (courtesy of Dr. Erwin A. Galinski and Dr. Matthias Kurz, Institute of Microbiology & Biotechnology, University of Bonn, Germany). First strand 3′ RACE cDNAs were prepared with a high Tm oligo(dT)18 and 1 μl of this cDNA was used in a final volume of 50 μl for the first round PCR amplification with MITF CDS common region primer. Subsequently, successive, nested, splice variant-specific amplifications were performed in a 50 μl PCR volume using 1 μl of the primary enriched RT-PCR reaction with corresponding forward and reverse primers (Table S2). 2.6. DNA splice junction amplification Blood-derived genomic DNA was amplified to confirm splice variants of MITF (+) and (−) forms in MITF cDNAs. The Expand Long Range, dNTPack (Roche S.p.A., Milan, Italy) was used following the manufacturer's instructions, including 0.3–0.5 μM specific primers (see Table S2), 500 μM dNTP mix, 3% DMSO, 100–150 ng of genomic DNA and 3.5 U of Expand Long Range Enzyme mix in a final 50 μl PCR volume. PCR was performed as per kit protocol (Roche). The reference MITF genomic locus at the exon 6–intron (6)–exon 7 splice junction was amplified in comparison to the orthologous MITF gene alignment of humans, mice, cows, dogs and sheep (Oar_v3.1:NENSG00000187098: ENST00000352241 intron 6:KNOWN_protein_coding). 2.7. Expression of sheep MITF isoforms in skin To possibly detect differences between the MITF (+) and (−) cDNA transcript expressions, we performed RT-PCR amplification using three

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different sets of isoform-specific (+ and −) primers as summarized in Table S2. Total RNA of 1.5 μg of each animal was reverse transcribed into cDNA using 200 U PrimScript™ Reverse Transcriptase (Takara Biotech, Japan) and 50 pmol oligo(dT) modified primer in a 20 μl reaction volume, as described above. For the RT-PCR reference, constitutively expressed glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 252 bp) and 18S rRNA (132 bp) were used as an equal loading control. Primers specific to the housekeeping gene (HKG) were designed from the corresponding Ovis aries NCBI GenBank Accession Nos. (Table S2) and amplified with the same PCR conditions and cycle numbers. 2.8. Cloning and sequencing All the selected amplicons were gel purified using Nucleospin columns (Macherey-Nagel, Germany) and cloned by using the TA cloning system (pGEM®-T Easy, Promega; pCR®2.1 TOPO, Invitrogen; InsTAclone™, Fermentas and pSC-A, StrataClone-UA, Stratagene). Clones were screened by M13 colony PCR amplification and positive clones were sequenced by the commercial vendors (StarSEQ, Germany; BMR sequencing, Italy) with M13 forward and/or reverse primers, or sequenced with any one of the gene specific primer for deeper sequencing of the inserts when necessary.

2.9. Sequence data Our newly sequenced data of MITF can be accessed through NCBI GenBank Accession Nos. FJ623631–FJ623632 and JN208138–JN208147 (Table S1).

2.10. Sequence analysis Whole mammalian genome scanning was done to identify the homologous regions of sheep MITF cDNA transcript variants using Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA (Altschul et al., 1997), ENSEMBL release 60 (Flicek et al., 2010) and BLAT (Kent, 2002) searches, sequentially. Sequences were edited, translated using the BioEdit v.7.0.5.2 (Ibis Therapeutics, Carlsbad, CA, USA) (Hall, 1999) and DNASTAR 7 (http://www.dnastar.com/) software packages. The open reading frame (ORF) of the full-length MITF cDNAs was determined by DNASTAR 7 and ORF Finder at NCBI (www.ncbi.nlm.nih.gov/gorf/). The positions of exons and introns, and the structure of the translated sheep MITF protein, were determined in reference to the MITF gene structure of humans, mice, dogs, and cows (source: NCBI, Ensembl). ClustalW2 (Thompson et al., 1994) and MUSCLE (Edgar, 2004) programs were used to align the DNA and protein sequences.

2.11. Use of other computational tools and databases Sheep MITF transcripts were searched on chr. 22 of the Bos taurus (Btau_6.1), on chr. 20 of the Canis lupus familiaris (Build 3.1) and on chr. 19 of the O. aries (Annotation Release 100) chromosomal map using the NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/ projects/mapview/). The sequence similarity was visualized with the Circos table viewer (http://mkweb.bcgsc.ca/tableviewer/). MITF polyadenylation sites were predicted using the polyADQ web server (Tabaska and Zhang, 1999). Alternative splicing pattern of the sheep MITF transcripts was predicted using ENSEMBL release 60 (Flicek et al., 2010) and AceView (Thierry-Mieg and Thierry-Mieg, 2006). The TargetScan program Release 6.2 (Friedman et al., 2009) was used to locate potential sheep MITF 3′ UTR miRNA target sites from humans, mice, cows, and dogs.

3. Results 3.1. Characterization of the MITF cDNA coding sequence To examine the MITF variant(s) expressed in the skin of merino sheep, total RNAs from the skin were reverse transcribed and the synthesized single strand cDNAs were amplified by PCR. We initially carried out the cDNA coding (CDS) region amplification using a degenerate primer pair, MITFfwd1 and MITFrev1 (Table S2). RT-PCR primers were selected based on the mammalian nucleotide (nt) sequence alignment of the MITF mRNA/cDNA encompassed to the open reading frame (ORF) of ~986 bp amplicon commonly known as ‘MITF (+)/(−)’. The expected size of amplicons was cloned and sequenced. The BLASTN sequence analyses of the partial ~986 bp sequence revealed the product as MITF-M, sharing identity with humans (88%), mice (86%), cattle (95%), pigs (91%) and dogs (91%). Although we obtained the desired MITF fragment, the initial amplification was performed by degenerate primers. Hence, to characterize the full-length MITF-M cDNA we used specific primers deduced from the primary product. The following combinations of primer pairs MITFfwd2, MITFrev2; MITFfwd4, MITFrev4; MITFfwd4, MITFrev5; and MITFfwd5, MITFrev7 (Table S2) were used which correspond to the expected amplicon sizes (in bp) of 582, 966 (+)/948(−); 581(+)/563(−); and 458, respectively (Fig. 2). Sequence analysis by BLASTN, BLASTP and ClustalW2 revealed all the products to be sheep MITF-M/MITF-M (−) isoform, identical to the one described and documented in humans (92%), mice (86%), goats and cattle (99%), pigs (95%), dogs (92%) and other mammals (b 92%). Upon overlapping the four individual fragments, we obtained a total of 1595 nt MITF-M cDNA fragment. The CDS (1242 bp) including the stop codon (TAG), corresponds to 413 aa, commonly known as ‘MITFM (−) isoform’. The first 127 bp before the start codon ‘ATG’ and the final 226 bp after the stop codon at 1595 nt were characterized as 5′ UTR and 3′ UTR, respectively. In an effort to identify the possible single nucleotide polymorphisms (SNPs) in the CDS of MITF, we also explored the 1595 nt cDNA fragments from colored (black and brown) individuals and compared it to the uncolored (white) individual. We did not observe any differences in the CDS region. 3.2. Identification of the basic alternative splice variants of MITF In order to characterize the two basic splice variants of MITF with (+) and without (−) an 18 bp insertion in the CDS for the amino acids, ‘ACIFPT’, we designed a common primer pair, namely MITFfwd7, MITFrev8(±) (Table S2). The goal was to amplify both transcript variants (±) in single RT-PCR. Using the primer pair ‘MITFfwd7’ and ‘MITFrev8(±)’, the expected amplicon sizes of 144 bp in the presence (+) and 126 bp for the absence (−) of 18 bp insertion were detected (Fig. 3A). It is not surprising that we did not obtain the ‘(+)’ variant in our earlier amplification, since the expression of MITF mRNA with the 18 bp insert (+) appeared to be lower than the (−) variant. In other words, there exists a considerable difference in the mRNA expression level between these two transcript variants (+/−), which is further explained in the later section. Upon overlapping the 144 bp (+) fragment to the 1595 nt MITF-M (−) isoform initially characterized, we obtained the alternative MITFM transcript variant 1613 nt in length. Of which, 1260 bp represents the CDS including the stop codon (TAG), corresponding to 416 aa, and commonly known as ‘MITF-M (+) isoform’. Further, we also looked into possible SNPs in the 144 bp (+) fragment of MITF-M (+) isoform of uncolored (white) vs. colored (black and brown) individuals. We did not observe any difference in the CDS. Interestingly, comparison of the complete CDS MITF (−) with MITF (+) cDNA sequence analysis revealed a ‘C’ to ‘T’ transition at 22 bp just before the 18 bp insertion site corresponding to the amino acid P179 (proline; no change in aa). This might affect dimer formation or

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Fig. 2. RT-PCR analysis of the sheep MITF coding regions (CDS) from the skin. Gel pictures show stepwise cDNA amplification of full-length MITF-M cDNA. Schematic structure of sheep MITF cDNA and sites of the primers for PCR amplification is given right below. The start and stop codons are indicated in blue and red respectively. Here and in subsequent figures ‘Wh’ represents individual of white merino sheep and ‘NC’ represents PCR negative control. ‘M’ indicates the DNA size markers: M1 = Gene Ruler—100 bp; M2 = Gene Ruler—1 kb; Sizes of PCR products are shown to the right. Arrows indicate the appropriate size of MITF amplicons.

DNA binding (Yasumoto et al., 1998). This change in nucleotide position (at nt 537) was observed in all three individuals irrespective of the color.

3.3. Characterization of MITF splice junction from the genomic DNA To verify the alternative splicing (AS) event that resulted in the basic MITF mRNA transcript variants i.e., (+) and (−) form, we amplified the intervening sequence between two exons. The MITF genomic locus at the exon 6–intron (6)–exon 7 splice junction was determined in comparison to the orthologous MITF gene assembly of sheep (ENSG00000187098:ENST00000352241 intron 6), humans, mice, and dogs (source: Ensembl). A common CDS forward primer, ‘MITFfwd7’ was designed on exon 6, and two different reverse primers were designed, namely ‘MITF-M(+)rev9’ and ‘MITF-M(−)rev10’ (Table S2) on exon 6–intron (6)–exon 7 boundaries of the human MITF gene. Both primer combinations were able to specifically amplify the expected length amplicons of 2525 bp for the MITF (+) form (Fig. 3B) and of 2507 bp for the MITF (−) form (data not shown) as verified by means of sequence analyses and orthologous comparison of these sheep MITF gene products to other mammals. The consensus ‘gtag’ intron (6) splice junction was covered in comparison to the orthologous MITF gene assembly of humans and mice. The overall similarity for the sheep MITF DNA splice region (+/−) in other vertebrates was found to be 80% in humans, 84% in mice, and 77% in dogs.

3.4. Expression of MITF (+) and (−) forms in skin To verify difference(s) between the expression level of the two basic splice variants of MITF i.e., (+) and (−), a common CDS primer, MITFfwd4 with two different reverse primers namely MITF-M(+)rev9 and MITF-M(−)rev10 (Table S2) was designed on the 18 bp insert spanning the exon 6–intron (6)–exon 7 boundaries (Ref. Seq. Sheep, Human, Mouse, Cow and Dog MITF gene). The corresponding primer combinations amplified a 292 bp for the (+) form and a 280 bp for the (−) form (Fig. 4A). The housekeeping genes (HKGs) GAPDH (252 bp) and 18SrRNA (132 bp) were used as an internal control (Fig. 4B). RT-PCR reactions produced fragments exhibiting clear differences in expression, as seen by band intensities between (+) vs. (−) form (Figs. 4A and 3A). Hence, we propose that the MITF gene expression at the mRNA transcript level is mediated via an intron-6 AS event in sheep.

3.5. Completion of 5′ and 3′ UTRs of sheep MITF and identification of two novel truncated isoforms To obtain the full length cDNAs, we performed the 5′ and 3′ RACE experiments sequentially. Different sets of primers (Table S2) were used for RACE RT-PCR amplification in order to ascertain the corresponding 5′ and 3′ untranslated regions (UTRs) of sheep MITF transcript variants.

Fig. 3. Analysis of the MITF basic transcript variants (+) and (−). Amplification strategy and sites of the primers for PCR amplification are given to the right. Panel A. Gel picture shows RTPCR electrophoretic pattern of products using MITF common CDS primers shown to the right. The amplification produced two fragments with different sizes, indicating the (+) and (−) for the 18 bp insertion. Panel B. Splice junction amplification of DNA covering (+) insertion. ‘M’ indicates DNA size markers: M1 = pBR322 DNA/AluI digest; M2 = λ-DNA EcoRI/HindIII digest. Sizes of PCR products are shown to the right as indicated by arrows.

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Fig. 4. Skin expression of sheep MITF basic isoform-specific (+) and (−). Amplification strategy and sites of the primers for PCR amplification are given to the right. The housekeeping genes (HKGs) GAPDH and 18SrRNA were used as an internal control. Panel A. Amplification of sheep MITF (+) and (−) splice variants. Panel B. Amplification of HKGs—GAPDH and 18SrRNA. Arrows indicate the appropriate size of MITF amplicons. ‘Br’, ‘Bl’, ‘Wh’ represent individual of brown, black and white merino sheep, respectively. ‘M’ indicates DNA size markers: M1 = Gene Ruler—100 bp and M2 = pBR322 DNA/AluI digest.

3.5.1. 5′ UTRs To determine the 5′ UTR of the sheep MITF, a gene-specific 5′ RACE cDNA was synthesized using the common CDS specific reverse primer ‘MITFrev8(±)’ (Table S2). Three μl of the dC-tailed cDNA was subjected to the first round RT-PCR amplification with the adapter forward primer ‘AAP’ and MITF gene specific primer ‘MITFrev2’ (Table S2). After the primary RT-PCR, a second round re-amplification was performed with using 10 μl of the primary enriched RT-PCR reaction. The secondary reaction yielded a complex pattern of amplicons ranging from ~200 to 1000 bp (Fig. 5A(a)). All the prominent 5′ RACE products were gel purified, cloned and sequenced. BLASTN results compared the sequenced clones of 827 bp, 395 bp and 222 bp (see Fig. 5A(a)) with the other mammalian MITF nucleotide sequences and were characterized as

sheep MITF isoforms. The rest of the amplicons were found to be nonspecific and omitted from further characterization. By overlapping and comparing the 5′ UTR sequence (827 bp) to the MITF common CDS region of 1242 bp including the stop codon (TAG), we obtained a complete CDS containing 1407 bp (of 1581 bp) which corresponds to 469 aa with a distinct N-terminus sequence, commonly known as the ‘MITF-E isoform’. 3.5.2. Discovery of novel truncated forms Overlapping the other two 5′ RACE products (395, 222 bp; Fig. 5A(a)) with the MITF common CDS region revealed two truncated N-terminus MITF isoforms covering a complete CDS containing 1059 bp (of 1151 bp) and 930 bp (of 973 bp) which corresponds to

Fig. 5. 5′ and 3′ RACE analysis of sheep MITF isoforms. Panel A. 5′ Rapid Amplification of cDNA (5′ RACE). The figure shows 5′ RACE-RT-PCR amplification of (a). MITF-E and two additional novel 5′ UTRs (from this study) namely MITF-Trn-1, 2. (b). MITF-M. (c). MITF-H. (d). MITF-B and (e). MITF-A. Arrows show 7 different MITF transcript variants including A, B, E, H, M, the counterparts of human MITF and the other two novel splice variants of MITF i.e., Trn-1, Trn-2 (N-termini, see Fig. 7) identified in this study. Panel B. 3′ Rapid Amplification of cDNA (3′ RACE). The corresponding arrows indicate the 3 different sizes of MITF 3′ UTR amplicons. ‘M’ indicates DNA size markers: M1 = Gene Ruler—1 kb, M2 = Gene Ruler—100 bp and M3 = 1 kb Gene Ruler. Schematic structure of sheep MITF cDNA and location of the primer sites for 5′ and 3′ RACE-PCR amplification is given below and to the right, respectively.

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353 aa and 310 aa, respectively. These two transcripts were found to be ‘novel’ and named as ‘MITF truncated forms-1, 2’ (in short Trn-1, 2; GenBank JN208143, JN208144; see Table S1). The result of this 5′ RACE RT-PCR analysis was further confirmed by two separate cDNA synthesis. 3.5.3. Other splice forms According to the AceView program (Thierry-Mieg and Thierry-Mieg, 2006), MITF transcription produces 16 different mRNAs. We knew that this gene is conserved across other mammalian species (Source: NCBI and Ensembl). Hence, we further extended the 5′ RACE experiment to understand the alternative splicing of MITF and subsequent transcript variants expressed in the skin of merino sheep. We tried 5′ RACE RTPCR several times with different combinations of primer pairs to evaluate expression of MITF 5′ ends. The design of primers and the 5′ RACE was difficult to execute further due to the increased complexity of MITF transcripts resulting from alternative splicing, as well as the unavailability of the sheep genome when the study was conducted. Henceforth, to evaluate the other MITF N-termini variants, we designed 5′ end MITF isoform-specific degenerate primers based on the alignment of other mammalian 5′ end MITF sequences. Also, it was difficult to PCR amplify the GC-rich 5′ end sequences. We solved this issue by either designing high Tm primers or following the use of homoectoine, a potent PCR enhancer (Schnoor et al., 2004) during the 5′ RACE cDNA synthesis protocol (Shi and Jarvis, 2006). Further, we identified four other MITF 5′ UTRs, commonly known as MITF-M (Fig. 5A(b)), MITF-H (Fig. 5A(c)), MITF-B (Fig. 5A(d)) and MITF-A (Fig. 5A(e)). The PCR primer pair and its subsequent specific amplicon sizes are given in Table S2. The GC contents for the identified MITF isoforms are 41% for B; 46% for E, H and M; 48% for Trn-1; 63% for Trn-2; and 72% for A determined by DNASTAR 7. Overlapping the sequenced clones of 582 bp, 193 bp, 193 bp and 191 bp with the MITF common CDS region resulted in four N-terminus MITF isoforms containing 1242 bp (of 1369 bp), 1515 bp (of 1594 bp), 1488 bp (of 1594 bp) and 1563 bp (of 1690 bp) which correspond to 414 aa, 505 aa, 496 aa and 521 aa, respectively, commonly known as MITF-M, MITFH, MITF-B and MITF-A. The expression of sheep isoforms MITF-H and MITF-A was also confirmed in the common CDS cDNA synthesis preparation (Fig. S2), which we used to amplify the initial ~ 986 bp MITF amplicon (see text above). We did not detect the other isoform MITFC, which has been previously reported to be undetectable in melanocyte lineages (Fuse et al., 1999). Overall, seven sheep MITF splice variants (Fig. 5A), with distinct Nterminus sequences (Source: NCBI-BLAST) were determined by 5′ RACE. Five of them were found to be the counterparts of human MITFA, B, E, H and M with the identity of 98%, 97%, 96%, 97% and 97% respectively. 3.5.4. 3′ UTRs The expected size of MITF 3′ UTR sequence, with respect to other mammalian MITF mRNA species, corresponds to ~ 3.5 kbp (Source: Ensembl, NCBI GenBank). We synthesized 3′ RACE cDNAs with oligo(dT)18 modified as a reverse primer. PCR amplification was performed with the common CDS region forward primer, ‘MITFfwd6’ and oligo(dT)18 modified as a reverse primer (Table S2). For the first time, we obtained a complex pattern of 3′ RACE PCR amplification as shown in Fig. 5B. To our surprise, sequence analysis by BLASTN and ClustalW2 confirmed the presence of three different sizes of MITF 3′ UTRs i.e., 714 bp, 1172 bp and 3275 bp in the skin of merino sheep. The rest of the amplicons were found to be non-specific and omitted from further evaluation. The 3′ RACE was repeated twice in two different cDNA preparations. In addition, this 3′ RACE PCR amplification was also confirmed in two other coat-colored (black, brown) individuals (data not shown) but this was not characterized further. At this stage, it was difficult to substantiate the existence of three different sizes of MITF 3′ UTR in the skin of merino sheep. In the case of longer 3′ UTR, one possible explanation could be

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that regulation of MITF gene expression involves microRNA (miRNA) target sites, which block translation (Fig. 6). After deducing the first 57 bp which corresponds to the common MITF CDS region, including the stop codon and the 32 bp adapter sequences from the oligo(dT)18 modified primer, we obtained the newly deduced MITF 3′ UTR as 625 bp (714 bp), 1083 bp (1172 bp) and 3186 bp (3275 bp), which includes the polyA nucleotides. To our knowledge, this is the first report describing the completeness of MITF 3′ UTR using 3′ RACE PCR. It was difficult to clone and sequence the entire 3′ UTR corresponding to 3275 bp, hence we used multiple nested primers to walk through the longest fragment. Using the default settings of NCBI, a BLASTN search was conducted with all three sheep MITF 3′ UTRs as independent query sequences. The sheep MITF 3′ UTR was highly conserved and found to share between 79% and 99% nucleotide identity with different mammalian representatives viz. goats (99%), cattle (95%), pigs (93%), dogs and horses (84–93%), humans (86–89%) and mice (80%). 3.6. Conservation of microRNA (miR) target sites A number of potential miRNA target sites are found within the longer 3′ UTR sequence of human MITF (Fig. 6A, B). However, in sheep, the potential miR sites that are located within the ~ 3.2 kbp MITF 3′ UTR sequence belong to the miR families of miR-25, miR-137, miR27abc/27a-3p, miR-148ab-3p/152 and miR-144 (Fig. 6B). The conservation of MITF 3′ UTR sequence in multiple species suggests the functional importance of these miRs in gene regulation (Hallsson et al., 2007). 3.7. Cross-species MITF amino acid sequence comparison Using the default settings of BLASTP, a search was conducted with the completed and translated sheep MITF protein sequences (from the present study) viz., 469 aa for MITF-E; 353 aa for MITF-Trn-1; 310 aa for MITF-Trn-2; 521 aa for MITF-A; 505 aa for MITF-H; 496 aa for MITF-B; 420 aa vs. 414 aa for MITF-M (+) and (−) as query sequences, respectively. BLASTP results revealed that amino acid sequences belonging to different mammalian representatives, including sheep MITF isoforms, were highly conserved. The percent identity of different MITF isoforms with other vertebrate species was found to be in the following sequence: cows (99–100%), goats (98–99%), horses (97–99%), dogs (96–99%), pigs (95–98%), cats (97–88%), humans (96–98%), mice and rats (91–93%). The graphical representation of evolutionary conservation of sheep MITF-M isoform is shown in Fig. S3. 3.8. Chromosome location and genomic structure of sheep MITF gene Upon scanning through the sheep genome for O. aries breed Texel at chromosome 19 (chr 19), covering position from 31,553 to 31,840 kb of Oar_v3.1 Genome Assembly, which encompasses a gene size of 287 kb for MITF. This represents that the gene length is between ~12 and 87% of the known MITF gene size, when compared to pigs (35.70 kb), cattle (53.47 kb), dogs (122.01 kb), mice (234.29 kb) and humans (248.90 kb) (source: Ensembl). The gene encoding the sheep MITF (NCBI gene ID: 101115163) is located within a syntenic group on chr 19 (Source: NCBI, Ensembl). This portion of sheep chr 19 is homologous to cattle chr 22, dog chr 20, pig chr 13, human chr 3, and mouse chr 6. Hence, a comparative chromosomal mapping was performed at the NCBI Map Viewer of the sheep MITF to cattle, pig and human MITF i.e., O.ari chr 19 to B.tau chr 22, S.sc chr 13 and H.s chr 3. The comparative map (Fig. S4) depicts the sheep MITF gene on chr 19 displaying regions of 31,553–31,840 kb (287 kb) covering counter-parts of cattle chr 22 (Bt RNA, B. taurus, 6.1), pig chr 13 (Ssc RNA, Sus scrofa, Sscrofa10.2) and human chr 3 (Hs RNA, Homo sapiens genome view Annotation Release 104 statistics). The complete structures of MITF isoforms, with their complexity and evolutionary sequence comparison, have been well represented and

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Fig. 6. Potential miRNA (miR) target sites on the sheep MITF 3′ UTRs. Panel A. Schematic representation of miR sites on the 3′ UTR sequences. Filled circle represents the three identified 3′ UTRs (in this study). Graphics on the 3′ UTR represent miR target sites conserved with other mammals. The distances are shown as base pairs (k) below the line indicating intervals of 1000 bp from exon 10. Panel B. Sequence conservation of the potential miR target sites with other mammals is depicted in the rectangular boxes.

documented for humans, mice and dogs (Udono et al., 2000; Shibahara et al., 2001; Hallsson et al., 2007; Tsuchida et al., 2009). Comparison of the MITF genomic DNA and cDNA sequence annotation with other mammalian sequence revealed that the sheep MITF gene consists of 10 exons interrupted by 9 introns (Fig. 7). The schematic representation of mRNA/cDNA structural architecture of sheep MITF expressed in the skin (from this study), with respect to human MITF, is shown in Fig. 7 and the AceView of human MITF gene maps on chr 3 is presented in Fig. S1.

4. Discussion Although considerable information on MITF cDNA sequences is available in the GenBank repository (NCBI) for other mammalian species (humans, chimpanzees, dogs, mice and other mammals), the fulllength mRNA (cDNA) structure for sheep (O. aries) remained unclear until December 2012 (source: PREDICTED, Oar_v3.1, GenBank, NCBI, December 2012). To our knowledge, there is no prior experimental evidence or report on the expression of sheep MITF in the skin or in any other tissue source. Taking into account the potential role exerted by MITF in melanogenesis (Steingrímsson et al., 2004) and coat pigmentation (Bismuth et al., 2005), sheep MITF cDNAs were amplified, cloned and sequenced from the skin of merino sheep (Figs. 2–5). Gene structure analysis has revealed that human, chimpanzee, dog and mouse MITF genes consist of nine alternative promoters. The consecutive first exons of these genes are separately spliced as 1M, 1H, 1A, 1B, 1C, 1D, 1E, 1J and 1mc. Downstream exons 2 to 10 are shared among all isoforms (Tsuchida et al., 2009; Ensembl; AceView). MITF isoform multiplicity with differential expression patterns, as well as functional diversity and redundancy, was explained previously (Fuse et al., 1999). This extensive alternative splicing (AS) event and conservation with other mammals has been shown in Fig. 1, Fig. S1 and Fig. S3, respectively. In this study, we have identified seven MITF transcript variants derived from sheep skin, including the two novel truncated transcripts viz., MITF-A, B, E, H, and M and Trn-1, 2 (Table S1) from the MITF gene (Fig. 7). Identified shortened isoforms (Trn-1, 2) may result as products of alternative splicing from an intron-1/exon-2 and intron-2/exon-3 region upstream of exon 4 of the sheep MITF gene

(Figs. 1 and 7). This scenario adds to the list of variants of the MITF gene that undergo alternative splicing (AS). Two basic MITF splice variants for the presence (+) or absence (−) of 18 bp insertion in the coding sequence (CDS) for the amino acids: ‘ACIFPT’ have been documented in humans, mice and other mammals (source: NCBI, Ensembl). The existence of (+) and (−) variants for the 18 bp insert has been reported in all of MITF isoforms expressed in adult dog tissues (Tsuchida et al., 2009). In contrast, human MITF-A, MITF-C, MITF-H and MITF-D lack this insertion (source: NCBI, Ensembl). In the present study, we were able to amplify the basic 18 bp insert region using cDNA (Figs. 3A and 4A) and then with gDNA (Fig. 3B). Until now, in sheep, the existence of exon 6 AS event in other MITF isoforms remains unknown. Amplification of MITF gene expression at the mRNA transcript level via an intron-6 AS event represents a significant difference between the MITF (+) and (−) variant (Figs. 3A and 4A). Similar observations have been reported in dogs (Tsuchida et al., 2009). The known MITFregulated gene, tyrosinase, was transcribed more efficiently by expression of MITF (+) isoform compared to MITF (−) isoform (Hemesath et al., 1994; Murakami et al., 2007). Also, MITF (+) isoform has been shown to be a strong inhibitor of DNA synthesis as demonstrated by BrdU incorporation analysis in vitro (Bismuth et al., 2005). This suggests that the 18 bp insertion has a widespread role in the regulation of MITF functions (Tsuchida et al., 2009). Usage of different promoters, giving rise to protein products with different N-termini (Figs. 5A and 7) has been demonstrated to be important for tissue-specific expression and can affect the transcriptional activation potential (Udono et al., 2000; Takemoto et al., 2002). Moreover, MITF mRNA (cDNA) structures (Fig. 7 and Fig. S1) and expression have been identified in a variety of other tissues such as skin (seen 43 times), melanocyte (21), kidney (17), uterus (17), breast (12) and 71 other tissues (53). This gene is expressed at high levels in a wide range of tissues (Source: AceView) revealing a heterogeneous expression profile of MITF. In the present study, three different sizes of 3′ UTRs were identified by 3′ RACE RT-PCR (Fig. 5B). The conservation of non-coding sequences in the sheep MITF 3′ UTR variants suggests that the 3′ UTRs might have a functional role in gene regulation. A number of miRNA (miR) target sequences are highly conserved in the 3′ UTR of MITF, but very little is

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Fig. 7. Architecture of the sheep MITF gene on chr. 19 (Oar_v3.1: ENSOARG00000009833). The homologous regions of the human MITF (Hu chr. 3 248.90 kb) are presented on top. Seven skin expressing sheep mRNAs are presented under the human gene structure. The direction of transcription is indicated by an arrow (from left to right) on Hu chr. 3. Alternative 5′ end splice variants (protein coding, N-termini) in the sheep MITF gene are indicated by open boxes with a start codon (ATG, blue) and its human counter parts are indicated by vertical black lines upstream of exon 2 (from A to B) on Hu chr. 3. Exons 2 to 10 (closed boxes) are common to all isoforms except for the last two novel variants identified in this study. The numbers on top of the boxes indicate exons which are interrupted by its corresponding introns (also numbered). Intronic distances between exons are given as base pairs (k) in comparison to human and sheep MITF gene (Ensembl). The stop codon (TAG) is indicated in red and the 3′ end variants are shown in light blue.

known about the biological function of specific MITF specific miRs. miRs, which anneal to the 3′ UTR of mRNAs in a sequence-specific fashion, either block translation or promote transcript degradation in posttranscriptional regulation of gene expression (Bartel, 2009; Kusenda et al., 2006). Two miRs namely miR-25 and miR-137 (Fig. 6) have been demonstrated as downregulators in the regulation of gene expression linked to coat color by targeting the transcription factor MITF in alpaca (Zhu et al., 2010) and a mouse model (Dong et al., 2012), respectively. A recent study on the pigmentation phenotype in Finnsheep using genome wide association study (GWAS) has shown strong evidence of three candidate genes (TYRP1, MITF and ASIP) associated with sheep coat color and patterns (M.H. Li et al., 2013). In other studies, the evolution of duplicated mitf genes (M. Li et al., 2013) and its alternative transcription generates multiple MITF isoforms in medaka fish (M. Li et al., 2014), with differential expression patterns and activities. The possible functional role(s) of these identified MITF isoforms including the two novel transcripts viz., MITF Trn-1, 2 and the existence of these two variants in different tissue sources in sheep remain unknown. Further studies are required to characterize the expression of these newly defined transcripts at the protein level in skin and its expression in other tissue sources, as well across other vertebrate species.

5. Conclusions In summary, we have reported the expression of sheep MITF-A, B, E, H, and M, the counterparts of human, mouse and dog MITF isoforms and three 3′ UTRs of sheep MITF transcript(s) from skin. In addition, we have identified two novel isoforms with truncated N-termini, which are encoded from a region within intron-1/exon-2 and intron-2/exon-3 boundaries and are expressed in skin. The sheep MITF isoforms are highly homologous to the nucleotide and the deduced amino acid sequences in humans and other mammals (NCBI-BLAST, Ensembl). It is important to understand the potential interacting partners with these unique Nterminal domains and bHLH-LZ domains that modulate the function of MITF isoforms, especially regulation of MITF-M by multiple signals (as reviewed in Shibahara et al., 2001). Although further work will be needed to characterize identified splice variants in other tissues where MITF is expressed, our data further describe the isoform multiplicity of MITF in sheep. Funding This in-house project on ‘Molecular Characterization of Pigmentation Genes in Merino Sheep’ was supported by the Faculty Research Grant to Dr. Antonietta La Terza and Dr. Carlo Renieri from the University of

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Camerino (Grant number: 20/2004), Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors' contributions ALT and CR conceived the project. ALT and SAS designed the study. SAS and DP performed the experiments. SAS analyzed the data and wrote the manuscript. ALT and CR contributed reagents/materials/analysis tools. ALT supervised the study and reviewed the manuscript. SAS, DP, CR and ALT contributed to the manuscript preparation, and approved its final version. Competing interests The authors declare that they have no competing interests. Acknowledgments The authors thank Faculty Research Grant, School of Environmental and Natural Sciences and School of Advanced Studies (SAS), University of Camerino, Italy for the research and technical support. We thank Profs. Dr. Erwin A. Galinski and Dr. Matthias Kurz of the Institute of Microbiology and Biotechnology, University of Bonn, Germany for generously providing the PCR additive ‘homoectoine’. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.09.031. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Amiel, J., Watkin, P.M., Tassabehji, M., Read, A.P., Winter, R.M., 1998. Mutation of the MITF gene in albinism–deafness syndrome (Tietz syndrome). Clin. Dysmorphol. 7, 17–20. Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Baxter, L.L., Hou, L., Loftus, S.K., Pavan, W.J., 2004. Spotlight on spotted mice: a review of white spotting mouse mutants and associated human pigmentation disorders. Pigment Cell Res. 17, 215–224. Bennett, D.C., Lamoreux, M.L., 2003. The color loci of mice: a genetic century. Pigment Cell Res. 16, 333–344. Bentley, N.J., Eisen, T., Goding, C., 1994. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol. Cell. Biol. 14, 7996–8006. Beraldi, D., McRae, A.F., Gratten, J., Slate, J., Visscher, P.M., Pemberton, J.M., 2006. Development of a linkage map and mapping of phenotypic polymorphisms in a free-living population of Soay sheep (Ovis aries). Genetics 173, 1521–1537. Bharti, K., Liu, W., Csermely, T., Bertuzzi, S., Arnheiterm, H., 2008. Alternative promoter use in eye development: the complex role and regulation of the transcription factor MITF. Development 135, 1169–1178. Bismuth, K., Maric, D., Arnheiter, H., 2005. MITF and cell proliferation: the role of alternative splice forms. Pigment Cell Res. 18, 349–359. Dong, C., Wang, H., Xue, L., Dong, Y., Yang, L., Fan, R., Yu, X., Tian, X., Ma, S., Smith, G.W., 2012. Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model. RNA 18, 1679–1686. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Flicek, P., Aken, B.L., Ballester, B., Beal, K., Bragin, E., et al., 2010. Ensembl's 10th year. Nucleic Acids Res. 38, D557–D562. Friedman, R.C., Farh, K.K.H., Burge, C.B., Bartel, D.P., 2009. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105. Fuse, N., Yasumoto, K., Takeda, K., Amae, S., Yoshizawa, M., Udono, T., Takahashi, K., Tamai, M., Tomita, Y., Tachibana, M., Shibahara, S., 1999. Molecular cloning of cDNA encoding a novel microphthalmia-associated transcription factor isoform with a distinct amino terminus. J. Biochem. (Tokyo) 126, 1043–1051. Goding, C.R., 2000. Mitf from neural crest to melanoma: signal transduction and transcription in the melanocyte lineage. Genes Dev. 14, 1712–1728. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hallsson, J.H., Favor, J., Hodgkinson, C., Glaser, T., Lamoreux, M.L., Magnúsdóttir, R., Gunnarsson, G.J., Sweet, H.O., Copeland, N.G., Jenkins, N.A., Steingrímsson, E., 2000.

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