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Analysis of horse myostatin gene and identification of single nucleotide polymorphisms in breeds of different morphological types.

Dall'Olio S, Fontanesi L, Nanni Costa L, Tassinari M, Minieri L, Falaschini A - J. Biomed. Biotechnol. (2010)

Bottom Line: Myostatin (MSTN) is a negative modulator of muscle mass.Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2.The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed).

View Article: PubMed Central - PubMed

Affiliation: DIPROVAL, Sezione di Allevamenti Zootecnici, Faculty of Agriculture, University of Bologna, 42123 Reggio Emilia, Italy.

ABSTRACT
Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

Show MeSH
Sequence analysis of the proximal promoter region and 5′-UTR of MSTN gene. Approximately 670 bp of the MSTN 5′-flanking region and 5′-UTR from horse, cattle, goat, human, mouse, pig, and sheep is shown. The consensus for transcription factor binding motives is shown in bold text and underlined. Numbering of the horse sequence is relative to the ATG start codon. Numbering of E-boxes is relative to the horse sequence. The two identified SNPs of horse MSTN gene are indicated with Y (T or C according to the IUPAC nomenclature) and in red.
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Related In: Results  -  Collection


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fig3: Sequence analysis of the proximal promoter region and 5′-UTR of MSTN gene. Approximately 670 bp of the MSTN 5′-flanking region and 5′-UTR from horse, cattle, goat, human, mouse, pig, and sheep is shown. The consensus for transcription factor binding motives is shown in bold text and underlined. Numbering of the horse sequence is relative to the ATG start codon. Numbering of E-boxes is relative to the horse sequence. The two identified SNPs of horse MSTN gene are indicated with Y (T or C according to the IUPAC nomenclature) and in red.

Mentions: Sequenced fragments of the horse MSTN gene were assembled into one sequence of 5724 bp (submitted to GenBank under accession number GQ183900) that resulted 100% identical with that that was, in the meantime, annotated in the EquCab2 horse genome assembly derived from a Thoroughbred horse (http://www.ensembl.org/Equus_caballus/Search/, Ensembl release 52-Dec 2009). Our sequence contained 671 bp upstream from the ATG start codon, 538 bp of the promoter, and the entire 5′-untranslated region (UTR) of 133 bp, the three exons (except 33 bp of exon 1), the two intervening introns, and 80 bp of the 3′-UTR (Figure 2). The transcription start site of the first exon was deduced from human and bovine MSTN exon 1 sequences [4, 6]. The coding regions of exons 1, 2, and 3 of the horse MSTN gene contained 373, 374, and 381 bp, respectively. Introns 1 and 2 included 1829 bp and 2016 bp, respectively, almost the same length reported in cattle (1840 bp and 2033 bp, respectively) and pig (1809 bp and 1980 bp, respectively) [6, 8]. Intron 1 is a type 1 intron as it interrupts a codon between the first and second exon whereas intron 2 is a type 0 intron as it divides the coding sequence between two codons as in other species [6, 8]. The analysed proximal promoter region and the 5′-UTR of the horse MSTN exhibited a degree of identity with the corresponding regions of other species ranging from 77% (mouse) to 90% (pig). Putative consensus DNA sequences known as transcription factor binding sites, DNA-binding motifs, or cis-regulatory elements were identified in the positive strand of horse promoter (Figure 3). Considering the general transcription factors, three different putative TATA boxes (TATA-1, TATA-2, and TATA-3) and one CCAAT box were detected. Among muscle-specific transcription factors, four E-boxes (named E1, E2, E3, and E4 boxes, Figure 3), one putative site for myocyte specific enhancer factor 2 (MEF2 or MEB1) and consensus sequences for FoxO and SMAD binding sites (CAAAATA and CAGACA, respectively) family sequences were identified. The alignment of MSTN promoter sequences across different species (horse, cattle, goat, human, mouse, pig, and sheep) revealed that these DNA-binding motives, particularly close to the TATA-1 surrounding sequence, were highly conserved across species. In particular, TATA-1 was conserved in all examined species except mouse, the second TATA sequence (TATA-2) was conserved across all seven species, and TATA-3 was conserved in all species except pig and mouse. The MEF2 and E-boxes were conserved in all the considered mammals except in human and mouse for E-box4. The E-boxes can be activated by the myogenic regulatory factors (MRFs: MyoD, Myf5, myogenin, and MRF4). MyoD upregulates MSTN transcription [33] and at the same time MSTN inhibits MyoD expression and activity regulating the differentiation of myoblasts into myotubes [34]. MyoD and MRF4 play competitive roles in myogenesis and might act as molecular switches to determine myogenic differentiation and cell proliferation, respectively [35]. Additional E-boxes were identified in the analysed region (such as an E-box located near the TATA-2 in pig and an additional E-box in all mammalian but cattle) and in the distal region of the promoter of the other mammals (not included in Figure 3). In cattle, Spiller et al. [33] showed the importance of three functional E-boxes (E3, E4, and E6) of which the E6, occupied by MyoD in vitro and in vivo, resulted crucial for the MSTN promoter activity. The close position of functional E-boxes suggests that they might function as a cluster to better sustain the stability of DNA-protein. Across the MSTN promoter sequences of all considered livestock species we identified the conserved position of sites matching the consensus for FoxO binding and the adjacent SMAD box whose presence was not evidenced in previous works [8, 33, 36]. Recent data demonstrated that these factors appear to act through independent pathways but additively to regulate the expression of MSTN and contribute to control muscle cell growth and differentiation [37, 38]. In addition, FoxO transcription factors plays a critical role in development of muscle atrophy by stimulating proteolysis and by increasing myostatin expression. Putative E-boxes were identified both in intron 1 (six boxes) and in intron 2 (six boxes) and one putative E-box was located in the 3′-UTR at seven nucleotides downstream of the TGA stop codon (data not shown). The presence of E-boxes in the introns and 3′-UTR of equine MSTN gene has not been described yet even if their occurrence has been highlighted recently in introns of porcine MSTN gene [8].


Analysis of horse myostatin gene and identification of single nucleotide polymorphisms in breeds of different morphological types.

Dall'Olio S, Fontanesi L, Nanni Costa L, Tassinari M, Minieri L, Falaschini A - J. Biomed. Biotechnol. (2010)

Sequence analysis of the proximal promoter region and 5′-UTR of MSTN gene. Approximately 670 bp of the MSTN 5′-flanking region and 5′-UTR from horse, cattle, goat, human, mouse, pig, and sheep is shown. The consensus for transcription factor binding motives is shown in bold text and underlined. Numbering of the horse sequence is relative to the ATG start codon. Numbering of E-boxes is relative to the horse sequence. The two identified SNPs of horse MSTN gene are indicated with Y (T or C according to the IUPAC nomenclature) and in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913906&req=5

fig3: Sequence analysis of the proximal promoter region and 5′-UTR of MSTN gene. Approximately 670 bp of the MSTN 5′-flanking region and 5′-UTR from horse, cattle, goat, human, mouse, pig, and sheep is shown. The consensus for transcription factor binding motives is shown in bold text and underlined. Numbering of the horse sequence is relative to the ATG start codon. Numbering of E-boxes is relative to the horse sequence. The two identified SNPs of horse MSTN gene are indicated with Y (T or C according to the IUPAC nomenclature) and in red.
Mentions: Sequenced fragments of the horse MSTN gene were assembled into one sequence of 5724 bp (submitted to GenBank under accession number GQ183900) that resulted 100% identical with that that was, in the meantime, annotated in the EquCab2 horse genome assembly derived from a Thoroughbred horse (http://www.ensembl.org/Equus_caballus/Search/, Ensembl release 52-Dec 2009). Our sequence contained 671 bp upstream from the ATG start codon, 538 bp of the promoter, and the entire 5′-untranslated region (UTR) of 133 bp, the three exons (except 33 bp of exon 1), the two intervening introns, and 80 bp of the 3′-UTR (Figure 2). The transcription start site of the first exon was deduced from human and bovine MSTN exon 1 sequences [4, 6]. The coding regions of exons 1, 2, and 3 of the horse MSTN gene contained 373, 374, and 381 bp, respectively. Introns 1 and 2 included 1829 bp and 2016 bp, respectively, almost the same length reported in cattle (1840 bp and 2033 bp, respectively) and pig (1809 bp and 1980 bp, respectively) [6, 8]. Intron 1 is a type 1 intron as it interrupts a codon between the first and second exon whereas intron 2 is a type 0 intron as it divides the coding sequence between two codons as in other species [6, 8]. The analysed proximal promoter region and the 5′-UTR of the horse MSTN exhibited a degree of identity with the corresponding regions of other species ranging from 77% (mouse) to 90% (pig). Putative consensus DNA sequences known as transcription factor binding sites, DNA-binding motifs, or cis-regulatory elements were identified in the positive strand of horse promoter (Figure 3). Considering the general transcription factors, three different putative TATA boxes (TATA-1, TATA-2, and TATA-3) and one CCAAT box were detected. Among muscle-specific transcription factors, four E-boxes (named E1, E2, E3, and E4 boxes, Figure 3), one putative site for myocyte specific enhancer factor 2 (MEF2 or MEB1) and consensus sequences for FoxO and SMAD binding sites (CAAAATA and CAGACA, respectively) family sequences were identified. The alignment of MSTN promoter sequences across different species (horse, cattle, goat, human, mouse, pig, and sheep) revealed that these DNA-binding motives, particularly close to the TATA-1 surrounding sequence, were highly conserved across species. In particular, TATA-1 was conserved in all examined species except mouse, the second TATA sequence (TATA-2) was conserved across all seven species, and TATA-3 was conserved in all species except pig and mouse. The MEF2 and E-boxes were conserved in all the considered mammals except in human and mouse for E-box4. The E-boxes can be activated by the myogenic regulatory factors (MRFs: MyoD, Myf5, myogenin, and MRF4). MyoD upregulates MSTN transcription [33] and at the same time MSTN inhibits MyoD expression and activity regulating the differentiation of myoblasts into myotubes [34]. MyoD and MRF4 play competitive roles in myogenesis and might act as molecular switches to determine myogenic differentiation and cell proliferation, respectively [35]. Additional E-boxes were identified in the analysed region (such as an E-box located near the TATA-2 in pig and an additional E-box in all mammalian but cattle) and in the distal region of the promoter of the other mammals (not included in Figure 3). In cattle, Spiller et al. [33] showed the importance of three functional E-boxes (E3, E4, and E6) of which the E6, occupied by MyoD in vitro and in vivo, resulted crucial for the MSTN promoter activity. The close position of functional E-boxes suggests that they might function as a cluster to better sustain the stability of DNA-protein. Across the MSTN promoter sequences of all considered livestock species we identified the conserved position of sites matching the consensus for FoxO binding and the adjacent SMAD box whose presence was not evidenced in previous works [8, 33, 36]. Recent data demonstrated that these factors appear to act through independent pathways but additively to regulate the expression of MSTN and contribute to control muscle cell growth and differentiation [37, 38]. In addition, FoxO transcription factors plays a critical role in development of muscle atrophy by stimulating proteolysis and by increasing myostatin expression. Putative E-boxes were identified both in intron 1 (six boxes) and in intron 2 (six boxes) and one putative E-box was located in the 3′-UTR at seven nucleotides downstream of the TGA stop codon (data not shown). The presence of E-boxes in the introns and 3′-UTR of equine MSTN gene has not been described yet even if their occurrence has been highlighted recently in introns of porcine MSTN gene [8].

Bottom Line: Myostatin (MSTN) is a negative modulator of muscle mass.Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2.The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed).

View Article: PubMed Central - PubMed

Affiliation: DIPROVAL, Sezione di Allevamenti Zootecnici, Faculty of Agriculture, University of Bologna, 42123 Reggio Emilia, Italy.

ABSTRACT
Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

Show MeSH