Gene 552 (2014) 165–175

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Alternative splicing of the sheepMITF gene: Novel transcripts detectablein 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

Abbreviations:MITF,microphthalmia-associated transccell growth factor receptor (SCFR)orproto-oncogenec-KitCD117; SCF, stem cell factor; bHLH-LZ, basic helix–loop–hsignaling protein; MC1R, melanocortin 1 receptor; TYRP1Tyr, tyrosinase; Dct/Tyrp-2, dopachrome-tautomeraseRACE-PCR, rapid amplification of cDNA ends-polymeraTranscription; dNTP, deoxyribonucleoside triphosphateDMSO, dimethyl sulfoxide; TdT, terminal deoxynudeoxycytidine triphosphate; GAPDH, glyceraldehyde 3-phribosomal RNA; RNA, ribonucleic acid;miRNA/miRs,micro⁎ Correspondence to: S.A. Saravanaperumal, Departmen

Engineering Enteric NeuroScience Program, Mayo Clinic, 28-98, Rochester, MN 55905, USA.⁎⁎ Corresponding author.

E-mail addresses: sivabiotech2002@yahoo.com (S.A. Sdario.pediconi@unicam.it (D. Pediconi), carlo.renieri@unicantonietta.laterza@unicam.it (A. La Terza).

http://dx.doi.org/10.1016/j.gene.2014.09.0310378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 January 2014Received in revised form 12 September 2014Accepted 15 September 2014Available online 18 September 2014

Keywords:Alternative splicing (AS)Exon skippingMerino sheepMicrophthalmia-associated transcription factor(MITF)Splice variant

Microphthalmia-associated transcription factor (MITF) is a basic helix–loop–helix leucine zipper (bHLH-LZ) tran-scription factor, which regulates the differentiation and development of melanocytes and pigment cell-specifictranscription of the melanogenesis enzyme genes. Though multiple splice variants of MITF have been reportedin humans, mice and other vertebrate species, in merino sheep (Ovis aries), MITF gene splicing has not yetbeen investigated until now. To investigate the sheep MITF isoforms, the full length mRNA/cDNAs from theskin of merino sheep were cloned, sequenced and characterized. Reverse transcriptase (RT)-PCR analysis andmolecular 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 bythe complete nucleotide sequencing of splice junction covering intron-6 (2463 bp),wherein an 18 bp intronic se-quence is retained into the CDS of MITF (+) isoform. Further, full-length cDNA libraries were enriched by themethod 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 mouseMITF, were identified by 5′ RACE. The other two 5′ RACE products were found to be novel splice variants ofMITF and represented as ‘MITF truncated form (Trn)-1, 2’. These alternative splice (AS) variants were illustratedusing comparative genome analysis. Bymeans of 3′ RACE three differentMITF 3′UTRs (625, 1083, 3167 bp)wereidentified and characterized.We also demonstrated that theMITF gene expression determined at transcript levelis mediated via an intron-6 splicing event. Here we summarize for the first time, the expression of seven MITFsplice variants with three distinct 3′ UTRs in the skin of merino sheep. Our data refine the structure of the MITFgene 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) pro-found, but remain mechanistically uncharacterized (Bennett andLamoreux, 2003). In sheep, the candidate genes for recessive black(ASIP) (Parsons et al., 1999a, 1999b), dominant black (MC1R) (Vage

ription factor; c-KIT,Mast/stemor tyrosine-proteinkinaseKit orelix leucine zipper; ASIP, Agouti, tyrosinase-related protein 1;/tyrosinase-related protein 2;se chain reaction; RT, Reverse; DEPC, diethyl pyrocarbonate;cleotidyl transferase; dCTP,osphate dehydrogenase; rRNA,RNAs; gDNA, genomicDNA.t of Physiology and Biomedical00 1st Street SW, Guggenheim

aravanaperumal),am.it (C. Renieri),

et al., 1999, 2003) and Brown (TYRP1) (Beraldi et al., 2006) have beenfound; these genes are known to influence pigmentation or pigmentsynthesis level. Many white spotting traits have been identified inmouse and man; and 10 of the responsible genes have been cloned(Baxter et al., 2004). It has been hypothesized that the gene for whitephenotype 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 Spot-ting) and stem cell factor (SCF, Steel), it is possible to obtain completelywhite live animals (Bennett and Lamoreux, 2003; Baxter et al., 2004;Hoekstra, 2006). In merino sheep, a novel splice variant of SCF gene in-volving premature termination of transcription has been reported(Saravanaperumal et al., 2012).

Microphthalmia-associated transcription factor (MITF) and itshuman counterpart huMITF (Tachibana et al., 1994) contain a basichelix–loop–helix-leucine zipper (bHLH-LZ) structure (Hodgkinsonet al., 1993; Hughes et al., 1993) required forDNAbinding anddimeriza-tion (Hallsson et al., 2000; Udono et al., 2000). MITF plays importantroles in thedevelopment and differentiation of neural crest-derivedme-lanocytes (Goding, 2000; Lekmine et al., 2007), bone marrow-derivedmast 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 coatcolor and microphthalmia (Tachibana et al., 1992), and leads to defectsin melanocytes, the retinal pigmented epithelium (RPE), mast cellsand osteoclasts (Hodgkinson et al., 1993; Widlund and Fisher, 2003;Steingrímsson et al., 2004). In humans, germline heterozygous muta-tions of the MITF gene are associated with the congenital pigmenta-tion/deafness condition, Waardenburg Syndrome (WS) type 2A(OMIM #193510) (Hughes et al., 1994; Tassabehji et al., 1994;Nobukuni et al., 1996), inwhich affected individuals display variable de-grees of pigmentation, and associated deafness due to melanocyte de-fects in the inner ear (Price and Fisher, 2001). A subtype of WS type2A, namely Tietz syndrome (OMIM #103500), is characterized by amore severe phenotype wherein affected patients display more severedeafness 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 en-zyme genes: tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1) anddopachrome-tautomerase (Dct/Tyrp-2) (Bentley et al., 1994; Yasumotoet al., 1994; Yasumoto and Shibahara, 1997).

Alternatively spliced transcript variants of humanMITF encodingdif-ferent isoformshave been identified. In humans,MITF consists of at leasteight 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, mouseand dogMITF genes contain multiple promoters and first exons, gener-ating multiple MITF isoforms, suggesting the functional diversity ofthese isoforms in a variety of tissues. Each isoform of MITF is differenti-ated by its unique N-terminus encoded by separatefirst exons designat-ed as 1M, 1H, 1A, 1B, 1C, 1D, 1E and 1J. All the isoforms ofMITF share the

Fig. 1. Schematic representation of the sevenMITF splice variants detected in the skin of sheep.alternative splice variants. In the above picture, the alternative splicing (AS)mechanismof the sto symbols). BLASTN and BLASTP analyses revealed that seven different N-termini of sheepMITF3 out of 4 validated alternative poly-adenylation sites (see the notation) were also identified b

entire downstream region containing a transactivation domain and abHLH-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 allowfor further studies into the regulation of gene expression in the sheepskin biology. Although many isoforms have been described for MITFgenes in humans, mice, and dogs, there is not currently experimentalevidence in sheep for the MITF transcript profile, especially in the skin.Very recently an updated version of this gene became available forsheep in NCBI (Gene ID: 101115163, PREDICTED version Oar_v3.1 up-dated on 03-June-2014). There exist two partial uncharacterized entriesone each for mRNA (EU028312.1) and DNA (GAAI01000006.1, from theheart). 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 callusand endometrium. To our knowledge this is the first experimental evi-dence of the completeMITF transcript profile detectable in the skin frommerino sheep.

In an effort, to better characterize themRNA/cDNA structure ofMITF inthe skin of merino sheep we performed cDNA and gDNA cloning,sequencing, and gene expressionbyRT-PCR analysis. In this study,we iso-lated and characterized sevenMITF splice variants from the skin that codefor distinct N-terminus sequences (Fig. 1). We isolated two novel splicevariants ofMITF, designated as ‘MITF truncated form (Trn)-1, 2’. The twobasic splice variants of MITF transcript variants, ‘(+) and (−)’, the com-monly known homologs of MITF in other mammals, are also presentedhere. We also demonstrated that the relative expression of MITF ‘(+)and (−)’ mRNA in the skin is mediated by an intron-6 splicing event. Inaddition, three different sizes ofMITF 3′UTRswere identified and charac-terized. This manuscript discusses the complete structural variation ofMITF mRNA (Fig. 7), and putative AS events on the intron-1/2 of theMITF gene by comparative genomics. This work provides a basis for un-derstandingMITF functions in the skin of sheep.

Translated sheepMITF cDNA sequences aligned with reference to humanMITF revealed 7heepMITF gene is depictedwith the possible splice donor and acceptor site (follow the keycDNA, identified by 5′ RACE (see Fig. 5A) are shownwith corresponding AS notation. Also,y 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), andimmediately frozen in liquid nitrogen until RNA extraction. Blood sam-ples were collected from the jugular vein of the same individuals withPAXgene Blood DNA Tubes (PreAnalytix/Qiagen) via a standard phle-botomy technique, processed immediately in the lab according to themanufacturer's protocol for the DNA isolation; DNA aliquots werestored at −80 °C. Samples were collected and recorded according tothe farm technicians from the following farms: “La Campana” locatedin the proximity of Montefiore dell'Aso (Ascoli Piceno, Marche) and“La Meridiana” in Umbertide (Perugia, Umbria), Italy.

2.2. Ethics statement

In agreement with the new European Directive on the protectionof animals used for scientific purposes (Directive 2010/63/EU, Article15, Annex VIII), all animal procedures used in the study are classifiedas ‘mild’ (i.e. procedures with no significant impairment of the well-being or general condition of the animals) and were preemptivelyapproved by the Animal Ethics Committee of the University ofCamerino.

2.3. RNA and DNA isolation and quantification

Total RNAs were extracted from the stored skin biopsies using TRIReagent (Sigma-Aldrich, Italy) according to the manufacturer's instruc-tions and treated with RNase-free DNase (Fermentas, Italy) to removecontaminated DNAs. Genomic DNAs were isolated from the blood sam-ples with the PAXgene Blood DNA kit (PreAnalytix/Qiagen) followingthe given handbook protocol. Qualitative assessments of the purifiedDNAs and RNAs were determined utilizing the Genesys 10 UV Spectro-photometer (Thermo Electron Corporation, Madison, USA).

2.4. cDNA synthesis and RT-PCR amplification

cDNAs were synthesized from total RNA extracted from the skin ofthe merino sheep. Reverse Transcription (RT) from 1–1.5 μg of RNA ina total volume of 20 μl containing 50 pmol oligo(dT) (18-mer) oroligo(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™ ReverseTranscriptase (Agilent, Stratagene, Italy) according to themanufacturer'sinstructions. The reaction was incubated for 60 min at 42 °C and thenheated at 70 °C for 15 min, and cooled on ice. Subsequently, 0.5–0.7 μlof the first strand cDNA reaction was used for PCR amplification. Reac-tions 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 (seeTable S2), 20–30 ng/μl cDNA and 1.5 U of proofreading Easy-A High-Fidelity PCR Cloning Enzyme (Agilent, Stratagene, Italy). Three-step RT-PCR amplification was performed in a MyCycler™ Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, California, USA), with the followingprogram: initial denaturation at 95 °C for 3 min, 5 primary cycles of94 °C for 1 min, followed by an annealing temperature (Ta°C) 3–5 °Cbelow the melting temperature (Tm) of the gene specific primer, 72 °Cfor 1 min. This was followed by 25 consecutive cycles of 94 °C for15–30 s, annealing temperature (Ta°C) for 15–30 s, and 72 °C for20–30 s with a final extension at 72 °C for 10 min and a hold tempera-ture 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 acrossprimer sets and the expected size of amplicons (see Table S2 for details).

The primer pairs specific toMITF isoform (+/−), used for amplifica-tion of the open reading frame (ORF) corresponding to the ~986 bp ofthe sheepMITF cDNA, were designed based on the coding sequence ho-mology 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 genespecific primer pairs were deduced from the 986 bp cDNA codingsequence (CDS) fragment in order obtain the full-length cDNAs. Allthe designed primer pairs were checked with the online softwaretools (http://www.sigmaaldrich.com/configurator/servlet/DesignTool?prod_type=STANDARD; Kalendar, 2010) before making an order withSigma. The primers used in this study were synthesized and purchasedfrom Sigma-Aldrich, Italy.

2.5. Rapid amplification of cDNA ends (5′ and 3′ RACEs)

5′ and 3′ RACE experiments to isolate and determine the sheep full-lengthMITF 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 atotal volume of 20 μl containing 2–2.5 pmol MITF gene specific splicevariant primer(s) (Table S2), 0.5 mM dNTPs, 1× RT buffer, 20 U ofRNase inhibitor, 200 U of PrimScript™ Reverse Transcriptase (TakaraBiotech, Japan) and StrataScript™ Reverse Transcriptase (Agilent,Stratagene, Italy) according to the manufacturer's instructions. Thereaction was incubated for 60 min at 50 °C and then heated at 70 °Cfor 15 min, cooled on ice and stored at −20 °C. The primer combina-tions used to amplify different MITF splice variants (5′/N-terminus)are provided in Table S2. Forward adapter primer sequences wereretrieved from the 5′ RACE kit, Invitrogen, USA and synthesized bySigma-Aldrich, Italy. The PCR amplification was carried out as describedabove for 36 cycles and the cycling conditions (e.g., Ta and timing inter-val) varied with 5′ RACE primer sets; see Table S2 for details about theexpected size of amplicons. Occasionally, 0.67 M homoectoine wasused for GC-rich 5′ RACE (courtesy of Dr. Erwin A. Galinski andDr. Matthias Kurz, Institute of Microbiology & Biotechnology, Universityof Bonn, Germany).

First strand 3′ RACE cDNAs were prepared with a high Tmoligo(dT)18 and 1 μl of this cDNA was used in a final volume of 50 μlfor the first round PCR amplification with MITF CDS common regionprimer. Subsequently, successive, nested, splice variant-specific ampli-ficationswere performed in a 50 μl PCR volume using 1 μl of the primaryenriched RT-PCR reaction with corresponding forward and reverseprimers (Table S2).

2.6. DNA splice junction amplification

Blood-derived genomic DNA was amplified to confirm splice vari-ants of MITF (+) and (−) forms in MITF cDNAs. The Expand LongRange, dNTPack (Roche S.p.A., Milan, Italy) was used following themanufacturer's instructions, including 0.3–0.5 μM specific primers (seeTable S2), 500 μM dNTP mix, 3% DMSO, 100–150 ng of genomic DNAand 3.5 U of Expand LongRange Enzymemix in a final 50 μl PCR volume.PCR was performed as per kit protocol (Roche). The reference MITFgenomic locus at the exon 6–intron (6)–exon 7 splice junction wasamplified in comparison to the orthologous MITF gene alignment ofhumans, 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 theMITF (+) and (−) cDNAtranscript expressions, we performed RT-PCR amplification using three

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different sets of isoform-specific (+ and −) primers as summarized inTable S2. Total RNA of 1.5 μg of each animal was reverse transcribedinto cDNA using 200 U PrimScript™ Reverse Transcriptase (Takara Bio-tech, Japan) and 50 pmol oligo(dT) modified primer in a 20 μl reactionvolume, as described above. For the RT-PCR reference, constitutivelyexpressed glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 252 bp)and 18S rRNA (132 bp) were used as an equal loading control. Primersspecific to the housekeeping gene (HKG)were designed from the corre-spondingOvis ariesNCBI GenBank Accession Nos. (Table S2) and ampli-fied with the same PCR conditions and cycle numbers.

2.8. Cloning and sequencing

All the selected amplicons were gel purified using Nucleospincolumns (Macherey-Nagel, Germany) and cloned by using the TA clon-ing 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 positiveclones were sequenced by the commercial vendors (StarSEQ,Germany; BMR sequencing, Italy) with M13 forward and/or reverseprimers, or sequenced with any one of the gene specific primer fordeeper sequencing of the inserts when necessary.

2.9. Sequence data

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

2.10. Sequence analysis

Whole mammalian genome scanning was done to identify thehomologous regions of sheep MITF cDNA transcript variants usingBasic Local Alignment Search Tool (BLAST) at the National Centerfor Biotechnology Information (NCBI), Bethesda, Maryland, USA(Altschul et al., 1997), ENSEMBL release 60 (Flicek et al., 2010) andBLAT (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-lengthMITF 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 deter-mined in reference to the MITF gene structure of humans, mice,dogs, and cows (source: NCBI, Ensembl). ClustalW2 (Thompsonet al., 1994) and MUSCLE (Edgar, 2004) programs were used toalign the DNA and protein sequences.

2.11. Use of other computational tools and databases

SheepMITF 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 onchr. 19 of the O. aries (Annotation Release 100) chromosomalmap using the NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/). The sequence similarity was visualized withthe Circos table viewer (http://mkweb.bcgsc.ca/tableviewer/). MITFpolyadenylation sites were predicted using the polyADQ web server(Tabaska and Zhang, 1999). Alternative splicing pattern of the sheepMITF transcripts was predicted using ENSEMBL release 60 (Fliceket al., 2010) and AceView (Thierry-Mieg and Thierry-Mieg, 2006).The TargetScan program Release 6.2 (Friedman et al., 2009) wasused to locate potential sheep MITF 3′ UTR miRNA target sites fromhumans, 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 merinosheep, total RNAs from the skin were reverse transcribed and the syn-thesized single strand cDNAswere amplified by PCR.We initially carriedout the cDNA coding (CDS) region amplification using a degenerateprimer pair, MITFfwd1 and MITFrev1 (Table S2). RT-PCR primers wereselected based on the mammalian nucleotide (nt) sequence alignmentof the MITF mRNA/cDNA encompassed to the open reading frame(ORF) of ~986 bp amplicon commonly known as ‘MITF (+)/(−)’. Theexpected size of amplicons was cloned and sequenced. The BLASTNsequence analyses of the partial ~986 bp sequence revealed the productas MITF-M, sharing identity with humans (88%), mice (86%), cattle(95%), pigs (91%) and dogs (91%). Although we obtained the desiredMITF fragment, the initial amplification was performed by degenerateprimers. Hence, to characterize the full-length MITF-M cDNA we usedspecific primers deduced from the primary product. The following com-binations of primer pairs MITFfwd2, MITFrev2; MITFfwd4, MITFrev4;MITFfwd4, MITFrev5; and MITFfwd5, MITFrev7 (Table S2) were usedwhich correspond to the expected amplicon sizes (in bp) of 582, 966(+)/948(−); 581(+)/563(−); and 458, respectively (Fig. 2). Sequenceanalysis by BLASTN, BLASTP and ClustalW2 revealed all the products tobe sheep MITF-M/MITF-M (−) isoform, identical to the one describedand documented in humans (92%), mice (86%), goats and cattle (99%),pigs (95%), dogs (92%) and other mammals (b92%).

Upon overlapping the four individual fragments, we obtained a totalof 1595 nt MITF-M cDNA fragment. The CDS (1242 bp) including thestop codon (TAG), corresponds to 413 aa, commonly known as ‘MITF-M (−) isoform’. The first 127 bp before the start codon ‘ATG’ and thefinal 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 CDSofMITF, we also explored the 1595nt cDNA fragmentsfrom colored (black and brown) individuals and compared it to theuncolored (white) individual. We did not observe any differences inthe 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 aminoacids, ‘ACIFPT’, we designed a common primer pair, namely MITFfwd7,MITFrev8(±) (Table S2). The goal was to amplify both transcript vari-ants (±) 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 inour earlier amplification, since the expression of MITF mRNA with the18 bp insert (+) appeared to be lower than the (−) variant. In otherwords, there exists a considerable difference in the mRNA expressionlevel between these two transcript variants (+/−), which is furtherexplained in the later section.

Upon overlapping the 144 bp (+) fragment to the 1595 nt MITF-M(−) isoform initially characterized, we obtained the alternative MITF-M transcript variant 1613 nt in length. Of which, 1260 bp representsthe CDS including the stop codon (TAG), corresponding to 416 aa, andcommonly known as ‘MITF-M (+) isoform’. Further, we also lookedinto possible SNPs in the 144 bp (+) fragment of MITF-M (+) isoformof uncolored (white) vs. colored (black and brown) individuals. Wedid 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 bpjust before the 18 bp insertion site corresponding to the amino acidP179 (proline; no change in aa). This might affect dimer formation or

Fig. 2.RT-PCRanalysis of the sheepMITF coding regions (CDS) from the skin.Gel pictures show stepwise cDNAamplification of full-lengthMITF-McDNA. Schematic structure of sheepMITFcDNA 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’ repre-sents 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 PCRproducts are shown to the right. Arrows indicate the appropriate size of MITF amplicons.

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

3.3. Characterization of MITF splice junction from the genomic DNA

To verify the alternative splicing (AS) event that resulted in thebasic MITF mRNA transcript variants i.e., (+) and (−) form, we am-plified the intervening sequence between two exons. TheMITF geno-mic locus at the exon 6–intron (6)–exon 7 splice junction wasdetermined in comparison to the orthologous MITF gene assemblyof 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 primerswere designed, namely ‘MITF-M(+)rev9’ and ‘MITF-M(−)rev10’(Table S2) on exon 6–intron (6)–exon 7 boundaries of the humanMITF gene. Both primer combinations were able to specifically amplifythe expected length amplicons of 2525 bp for the MITF (+) form(Fig. 3B) and of 2507 bp for theMITF (−) form (data not shown) as ver-ified by means of sequence analyses and orthologous comparison ofthese sheep MITF gene products to other mammals. The consensus ‘gt-ag’ intron (6) splice junction was covered in comparison to theorthologous MITF gene assembly of humans and mice. The overall sim-ilarity for the sheepMITF DNA splice region (+/−) in other vertebrateswas found to be 80% in humans, 84% in mice, and 77% in dogs.

Fig. 3.Analysis of theMITF basic transcript variants (+) and (−). Amplification strategy and sitePCR electrophoretic patternof products usingMITF commonCDSprimers shown to the right. Thethe 18 bp insertion. Panel B. Splice junction amplification ofDNA covering (+) insertion. ‘M’ indiSizes of PCR products are shown to the right as indicated by arrows.

3.4. Expression of MITF (+) and (−) forms in skin

To verify difference(s) between the expression level of the two basicsplice variants of MITF i.e., (+) and (−), a common CDS primer,MITFfwd4 with two different reverse primers namely MITF-M(+)rev9and MITF-M(−)rev10 (Table S2) was designed on the 18 bp insertspanning the exon 6–intron (6)–exon 7 boundaries (Ref. Seq. Sheep,Human, Mouse, Cow and Dog MITF gene). The corresponding primercombinations amplified a 292 bp for the (+) form and a 280 bp forthe (−) 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 differ-ences in expression, as seen by band intensities between (+) vs. (−)form (Figs. 4A and 3A). Hence, we propose that the MITF gene expres-sion at the mRNA transcript level is mediated via an intron-6 AS eventin sheep.

3.5. Completion of 5′ and 3′ UTRs of sheep MITF and identification of twonovel truncated isoforms

To obtain the full length cDNAs, we performed the 5′ and 3′ RACEexperiments sequentially. Different sets of primers (Table S2) wereused for RACE RT-PCR amplification in order to ascertain the corre-sponding 5′ and 3′ untranslated regions (UTRs) of sheepMITF transcriptvariants.

s of the primers for PCR amplification are given to the right. Panel A. Gel picture shows RT-amplification produced two fragmentswith different sizes, indicating the (+)and (−) forcatesDNA sizemarkers:M1= pBR322DNA/AluI digest;M2=λ-DNAEcoRI/HindIII digest.

Fig. 4. Skin expression of sheepMITF basic isoform-specific (+) and (−). Amplification strategy and sites of the primers for PCR amplification are given to the right. The housekeepinggenes (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 and18SrRNA. Arrows indicate the appropriate size ofMITF amplicons. ‘Br’, ‘Bl’, ‘Wh’ represent individual of brown, black andwhitemerino sheep, respectively. ‘M’ indicates DNA sizemarkers:M1 = Gene Ruler—100 bp and M2 = pBR322 DNA/AluI digest.

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3.5.1. 5′ UTRsTo 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 cDNAwas subjectedto the first round RT-PCR amplificationwith the adapter forward primer‘AAP’ andMITF gene specific primer ‘MITFrev2’ (Table S2). After the pri-mary RT-PCR, a second round re-amplification was performed withusing 10 μl of the primary enriched RT-PCR reaction. The secondaryreaction yielded a complex pattern of amplicons ranging from ~200 to1000 bp (Fig. 5A(a)). All the prominent 5′ RACE products were gel puri-fied, cloned and sequenced. BLASTN results compared the sequencedclones of 827 bp, 395 bp and 222 bp (see Fig. 5A(a)) with the othermammalian MITF nucleotide sequences and were characterized as

Fig. 5. 5′ and 3′ RACE analysis of sheepMITF isoforms. Panel A. 5′ Rapid Amplification of cDNA (5novel 5′ UTRs (from this study) namelyMITF-Trn-1, 2. (b).MITF-M. (c).MITF-H. (d).MITF-B ancounterparts of human MITF and the other two novel splice variants of MITF i.e., Trn-1, Trn-2 (RACE). The corresponding arrows indicate the 3 different sizes of MITF 3′ UTR amplicons. ‘MM3 = 1 kb Gene Ruler. Schematic structure of sheepMITF cDNA and location of the primer site

sheep MITF isoforms. The rest of the amplicons were found to be non-specific and omitted from further characterization. By overlapping andcomparing the 5′ UTR sequence (827 bp) to the MITF common CDSregion of 1242 bp including the stop codon (TAG), we obtained a com-plete CDS containing 1407 bp (of 1581 bp)which corresponds to 469 aawith a distinct N-terminus sequence, commonly known as the ‘MITF-Eisoform’.

3.5.2. Discovery of novel truncated formsOverlapping the other two 5′ RACE products (395, 222 bp;

Fig. 5A(a)) with the MITF common CDS region revealed two truncatedN-terminus MITF isoforms covering a complete CDS containing1059 bp (of 1151 bp) and 930 bp (of 973 bp) which corresponds to

′ RACE). The figure shows 5′ RACE-RT-PCR amplification of (a).MITF-E and two additionald (e).MITF-A. Arrows show 7 differentMITF transcript variants including A, B, E, H, M, theN-termini, see Fig. 7) identified in this study. Panel B. 3′ Rapid Amplification of cDNA (3′’ indicates DNA size markers: M1 = Gene Ruler—1 kb, M2 = Gene Ruler—100 bp ands 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 cDNAsynthesis.

3.5.3. Other splice formsAccording to the AceView program (Thierry-Mieg and Thierry-Mieg,

2006), MITF transcription produces 16 different mRNAs. We knew thatthis gene is conserved across other mammalian species (Source: NCBIand Ensembl). Hence, we further extended the 5′ RACE experiment tounderstand the alternative splicing of MITF and subsequent transcriptvariants expressed in the skin of merino sheep. We tried 5′ RACE RT-PCR several times with different combinations of primer pairs to evalu-ate expression of MITF 5′ ends. The design of primers and the 5′ RACEwas difficult to execute further due to the increased complexity ofMITF transcripts resulting from alternative splicing, as well as the un-availability of the sheep genomewhen the studywas conducted.Hence-forth, to evaluate the otherMITFN-termini variants, we designed 5′ endMITF isoform-specific degenerate primers based on the alignment ofother mammalian 5′ end MITF sequences. Also, it was difficult to PCRamplify the GC-rich 5′ end sequences.We solved this issue by either de-signing high Tm primers or following the use of homoectoine, a potentPCR enhancer (Schnoor et al., 2004) during the 5′ RACE cDNA synthesisprotocol (Shi and Jarvis, 2006). Further, we identified four otherMITF 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 andits subsequent specific amplicon sizes are given in Table S2. The GC con-tents for the identifiedMITF 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 bpwith theMITF commonCDS region resulted in fourN-terminusMITF iso-forms 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, 505aa, 496 aa and 521 aa, respectively, commonly known asMITF-M,MITF-H, MITF-B and MITF-A. The expression of sheep isoforms MITF-H andMITF-A was also confirmed in the common CDS cDNA synthesis prepa-ration (Fig. S2), which we used to amplify the initial ~986 bp MITFamplicon (see text above). We did not detect the other isoform MITF-C, which has been previously reported to be undetectable inmelanocytelineages (Fuse et al., 1999).

Overall, seven sheep MITF splice variants (Fig. 5A), with distinct N-terminus sequences (Source: NCBI-BLAST) were determined by 5′RACE. Five of them were found to be the counterparts of human MITF-A, B, E, H and M with the identity of 98%, 97%, 96%, 97% and 97%respectively.

3.5.4. 3′ UTRsThe 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 areverse primer. PCR amplification was performed with the commonCDS region forward primer, ‘MITFfwd6’ and oligo(dT)18 modified as areverse primer (Table S2). For the first time,we obtained a complex pat-tern of 3′ RACE PCR amplification as shown in Fig. 5B. To our surprise,sequence analysis by BLASTN and ClustalW2 confirmed the presenceof three different sizes of MITF 3′ UTRs i.e., 714 bp, 1172 bp and3275 bp in the skin of merino sheep. The rest of the amplicons werefound to be non-specific and omitted from further evaluation. The 3′RACE was repeated twice in two different cDNA preparations. In addi-tion, this 3′ RACE PCR amplification was also confirmed in two othercoat-colored (black, brown) individuals (data not shown) but this wasnot characterized further. At this stage, it was difficult to substantiatethe existence of three different sizes ofMITF 3′UTR in the skin ofmerinosheep. In the case of longer 3′ UTR, one possible explanation could be

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 commonMITF CDS region, including the stop codon and the 32 bp adapter se-quences from the oligo(dT)18 modified primer, we obtained the newlydeduced MITF 3′ UTR as 625 bp (714 bp), 1083 bp (1172 bp) and3186 bp (3275 bp),which includes thepolyAnucleotides. To our knowl-edge, this is the first report describing the completeness ofMITF 3′ UTRusing 3′ RACE PCR. It was difficult to clone and sequence the entire 3′UTR corresponding to 3275 bp, hence we used multiple nested primerstowalk through the longest fragment. Using the default settings of NCBI,a BLASTN searchwas conductedwith all three sheepMITF 3′UTRs as in-dependent query sequences. The sheep MITF 3′ UTR was highly con-served and found to share between 79% and 99% nucleotide identitywith different mammalian representatives viz. goats (99%), cattle(95%), pigs (93%), dogs and horses (84–93%), humans (86–89%) andmice (80%).

3.6. Conservation of microRNA (miR) target sites

A number of potential miRNA target sites are found within the lon-ger 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, miR-27abc/27a-3p,miR-148ab-3p/152 andmiR-144 (Fig. 6B). The conserva-tion ofMITF 3′UTR sequence inmultiple species suggests the functionalimportance 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 withthe completed and translated sheep MITF protein sequences (from thepresent study) viz., 469 aa for MITF-E; 353 aa for MITF-Trn-1; 310 aafor MITF-Trn-2; 521 aa for MITF-A; 505 aa for MITF-H; 496 aa forMITF-B; 420 aa vs. 414 aa for MITF-M (+) and (−) as query sequences,respectively. BLASTP results revealed that amino acid sequences belong-ing to different mammalian representatives, including sheep MITF iso-forms, were highly conserved. The percent identity of different MITFisoformswith other vertebrate species was found to be in the followingsequence: cows (99–100%), goats (98–99%), horses (97–99%), dogs(96–99%), pigs (95–98%), cats (97–88%), humans (96–98%), mice andrats (91–93%). The graphical representation of evolutionary conserva-tion 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 atchromosome 19 (chr 19), covering position from 31,553 to 31,840 kb ofOar_v3.1 Genome Assembly, which encompasses a gene size of 287 kbfor MITF. This represents that the gene length is between ~12 and 87%of the knownMITF 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 cattlechr 22, dog chr 20, pig chr 13, human chr 3, and mouse chr 6. Hence, acomparative chromosomal mapping was performed at the NCBI MapViewer of the sheep MITF to cattle, pig and human MITF i.e., O.ari chr19 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 of31,553–31,840 kb (287 kb) covering counter-parts of cattle chr 22 (BtRNA, B. taurus, 6.1), pig chr 13 (Ssc RNA, Sus scrofa, Sscrofa10.2) andhuman chr 3 (Hs RNA, Homo sapiens genome view Annotation Release104 statistics).

The complete structures ofMITF isoforms, with their complexity andevolutionary sequence comparison, have been well represented and

Fig. 6. Potential miRNA (miR) target sites on the sheepMITF 3′UTRs. Panel A. Schematic representation ofmiR 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 of1000 bp from exon 10. Panel B. Sequence conservation of the potential miR target sites with other mammals is depicted in the rectangular boxes.

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documented for humans, mice and dogs (Udono et al., 2000; Shibaharaet al., 2001; Hallsson et al., 2007; Tsuchida et al., 2009). Comparison ofthe MITF genomic DNA and cDNA sequence annotation with othermammalian sequence revealed that the sheep MITF gene consists of10 exons interrupted by 9 introns (Fig. 7). The schematic representationof mRNA/cDNA structural architecture of sheep MITF expressed in theskin (from this study), with respect to human MITF, is shown in Fig. 7and the AceView of human MITF gene maps on chr 3 is presented inFig. S1.

4. Discussion

Although considerable information on MITF cDNA sequences isavailable in the GenBank repository (NCBI) for other mammalian spe-cies (humans, chimpanzees, dogs, mice and other mammals), the full-length mRNA (cDNA) structure for sheep (O. aries) remained unclearuntil December 2012 (source: PREDICTED, Oar_v3.1, GenBank, NCBI,December 2012). To our knowledge, there is no prior experimentalevidence or report on the expression of sheep MITF in the skin or inany other tissue source. Taking into account the potential role exertedbyMITF inmelanogenesis (Steingrímsson et al., 2004) and coat pigmen-tation (Bismuth et al., 2005), sheepMITF cDNAs were amplified, clonedand sequenced from the skin of merino sheep (Figs. 2–5). Gene struc-ture analysis has revealed that human, chimpanzee, dog and mouseMITF genes consist of nine alternative promoters. The consecutive firstexons 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 iso-forms (Tsuchida et al., 2009; Ensembl; AceView). MITF isoform multi-plicity with differential expression patterns, as well as functionaldiversity and redundancy, was explained previously (Fuse et al.,1999). This extensive alternative splicing (AS) event and conservationwith other mammals has been shown in Fig. 1, Fig. S1 and Fig. S3,respectively. In this study, we have identified sevenMITF transcript var-iants derived from sheep skin, including the two novel truncated tran-scripts viz., MITF-A, B, E, H, and M and Trn-1, 2 (Table S1) from theMITF gene (Fig. 7). Identified shortened isoforms (Trn-1, 2) may resultas products of alternative splicing from an intron-1/exon-2 andintron-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 MITFgene that undergo alternative splicing (AS).

Two basicMITF 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 forthe 18 bp insert has been reported in all of MITF isoforms expressed inadult dog tissues (Tsuchida et al., 2009). In contrast, human MITF-A,MITF-C,MITF-H andMITF-D lack this insertion (source: NCBI, Ensembl).In the present study, we were able to amplify the basic 18 bp insertregion using cDNA (Figs. 3A and 4A) and then with gDNA (Fig. 3B).Until now, in sheep, the existence of exon 6 AS event in otherMITF iso-forms remains unknown.

Amplification of MITF gene expression at the mRNA transcript levelvia an intron-6 AS event represents a significant difference betweenthe MITF (+) and (−) variant (Figs. 3A and 4A). Similar observationshave been reported in dogs (Tsuchida et al., 2009). The known MITF-regulated gene, tyrosinase, was transcribed more efficiently by expres-sion of MITF (+) isoform compared to MITF (−) isoform (Hemesathet al., 1994; Murakami et al., 2007). Also, MITF (+) isoform has beenshown to be a strong inhibitor of DNA synthesis as demonstrated byBrdU incorporation analysis in vitro (Bismuth et al., 2005). This suggeststhat the 18 bp insertion has a widespread role in the regulation ofMITFfunctions (Tsuchida et al., 2009).

Usage of different promoters, giving rise to protein products withdifferentN-termini (Figs. 5A and 7) has been demonstrated to be impor-tant for tissue-specific expression and can affect the transcriptionalactivation potential (Udono et al., 2000; Takemoto et al., 2002). More-over,MITFmRNA (cDNA) structures (Fig. 7 and Fig. S1) and expressionhave been identified in a variety of other tissues such as skin (seen 43times), melanocyte (21), kidney (17), uterus (17), breast (12) and 71other tissues (53). This gene is expressed at high levels in a widerange of tissues (Source: AceView) revealing a heterogeneous expres-sion profile of MITF.

In the present study, three different sizes of 3′ UTRs were identifiedby 3′ RACE RT-PCR (Fig. 5B). The conservation of non-coding sequencesin the sheepMITF 3′UTR variants suggests that the 3′UTRsmight have afunctional role in gene regulation. A number of miRNA (miR) targetsequences are highly conserved in the 3′ UTR of MITF, but very little is

Fig. 7. Architecture of the sheepMITF gene on chr. 19 (Oar_v3.1: ENSOARG00000009833). The homologous regions of the humanMITF (Hu chr. 3 248.90 kb) are presented on top. Sevenskin 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′ endsplice variants (protein coding, N-termini) in the sheepMITF gene are indicated by open boxes with a start codon (ATG, blue) and its human counter parts are indicated by vertical blacklines upstream of exon 2 (fromA 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 ontop of the boxes indicate exonswhich are interrupted by its corresponding introns (also numbered). Intronic distances between exons are given as base pairs (k) in comparison to humanand sheep MITF gene (Ensembl). The stop codon (TAG) is indicated in red and the 3′ end variants are shown in light blue.

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known about the biological function of specificMITF specificmiRs.miRs,which anneal to the 3′ UTR of mRNAs in a sequence-specific fashion, ei-ther block translation or promote transcript degradation in post-transcriptional regulation of gene expression (Bartel, 2009; Kusendaet al., 2006). Two miRs namely miR-25 and miR-137 (Fig. 6) havebeen demonstrated as downregulators in the regulation of gene expres-sion linked to coat color by targeting the transcription factorMITF in al-paca (Zhu et al., 2010) and a mouse model (Dong et al., 2012),respectively. A recent study on the pigmentation phenotype inFinnsheep using genome wide association study (GWAS) has shownstrong evidence of three candidate genes (TYRP1,MITF and ASIP) associ-ated with sheep coat color and patterns (M.H. Li et al., 2013). In otherstudies, the evolution of duplicated mitf genes (M. Li et al., 2013)and its alternative transcription generates multiple MITF isoforms inmedaka fish (M. Li et al., 2014), with differential expression patternsand activities.

The possible functional role(s) of these identified MITF isoformsincluding the two novel transcripts viz., MITF Trn-1, 2 and the exis-tence of these two variants in different tissue sources in sheepremain unknown. Further studies are required to characterize theexpression of these newly defined transcripts at the protein level inskin and its expression in other tissue sources, as well across othervertebrate species.

5. Conclusions

In summary, we have reported the expression of sheepMITF-A, B, E,H, andM, the counterparts of human,mouse and dogMITF isoforms andthree 3′UTRs of sheepMITF transcript(s) from skin. In addition,we haveidentified two novel isoforms with truncated N-termini, which areencoded from a region within intron-1/exon-2 and intron-2/exon-3boundaries and are expressed in skin. The sheepMITF isoforms are high-ly homologous to the nucleotide and the deduced amino acid sequencesin humans and other mammals (NCBI-BLAST, Ensembl). It is importantto understand the potential interacting partners with these unique N-terminal domains and bHLH-LZ domains that modulate the function ofMITF isoforms, especially regulation of MITF-M by multiple signals (asreviewed in Shibahara et al., 2001). Although furtherworkwill be need-ed to characterize identified splice variants in other tissues whereMITFis expressed, our data further describe the isoform multiplicity of MITFin sheep.

Funding

This in-house project on ‘Molecular Characterization of PigmentationGenes in Merino Sheep’ was supported by the Faculty Research Grant toDr. 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 instudy design, data collection and analysis, decision to publish, or prepa-ration 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 andwrote themanuscript. ALT and CR contributed reagents/materials/anal-ysis tools. ALT supervised the study and reviewed the manuscript. SAS,DP, CR and ALT contributed to the manuscript preparation, andapproved its final version.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

The authors thank Faculty Research Grant, School of Environmentaland Natural Sciences and School of Advanced Studies (SAS), Universityof Camerino, Italy for the research and technical support. We thankProfs. Dr. Erwin A. Galinski and Dr. Matthias Kurz of the Institute ofMicrobiology and Biotechnology, University of Bonn, Germany for gen-erously 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.

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