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Runx family genes in a cartilaginous fish, the elephant shark (Callorhinchus milii).

Nah GS, Lim ZW, Tay BH, Osato M, Venkatesh B - PLoS ONE (2014)

Bottom Line: Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3.Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci.Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The Runx family genes encode transcription factors that play key roles in hematopoiesis, skeletogenesis and neurogenesis and are often implicated in diseases. We describe here the cloning and characterization of Runx1, Runx2, Runx3 and Runxb genes in the elephant shark (Callorhinchus milii), a member of Chondrichthyes, the oldest living group of jawed vertebrates. Through the use of alternative promoters and/or alternative splicing, each of the elephant shark Runx genes expresses multiple isoforms similar to their orthologs in human and other bony vertebrates. The expression profiles of elephant shark Runx genes are similar to those of mammalian Runx genes. The syntenic blocks of genes at the elephant shark Runx gene loci are highly conserved in human, but represented by shorter conserved blocks in zebrafish indicating a higher degree of rearrangements in this teleost fish. Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3. Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci. Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu. In summary, our analysis reveals that the genomic organization and expression profiles of Runx genes were already complex in the common ancestor of jawed vertebrates.

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Exon-intron organization and protein sequence encoded by the elephant shark Runxβ.(A) Schematic representation of the genomic structure and the three transcripts cloned (CmRunxb types 1,2 and 3). Exons are indicated by boxes. The vertical dashed lines indicate internal splice sites located within a coding exon. The 5′ and 3′UTRs are represented as crosshatched boxes. (B) Alignment of elephant shark and human RUNXβ amino acid sequences using ClustalW. Conserved residues are shaded grey. Cm, Callorhinchus milii; Hs, Homo sapiens. (C) Expression patterns of elephant shark Runxb transcripts. Relative expression levels of Runxb Types 1+2 and Type 3 in various tissues of the elephant shark determined by qRT-PCR.
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pone-0093816-g009: Exon-intron organization and protein sequence encoded by the elephant shark Runxβ.(A) Schematic representation of the genomic structure and the three transcripts cloned (CmRunxb types 1,2 and 3). Exons are indicated by boxes. The vertical dashed lines indicate internal splice sites located within a coding exon. The 5′ and 3′UTRs are represented as crosshatched boxes. (B) Alignment of elephant shark and human RUNXβ amino acid sequences using ClustalW. Conserved residues are shaded grey. Cm, Callorhinchus milii; Hs, Homo sapiens. (C) Expression patterns of elephant shark Runxb transcripts. Relative expression levels of Runxb Types 1+2 and Type 3 in various tissues of the elephant shark determined by qRT-PCR.

Mentions: We cloned the full-length coding sequence of the elephant shark gene encoding the β-subunit of the CBF heterodimeric complex, Runxb. The exon-intron organization of elephant shark Runxb (CmRunxb) gene is identical to that of its human ortholog (Fig. 9A) [66]. Furthermore, CmRunxb is transcribed into at least three isoforms that are homologous to the human RUNXB type 1, 2 and 3 isoforms. The CmRunxb type 1, 2 and 3 isoforms encode proteins of 188, 185 and 156 amino acids, respectively (Fig. 9B). CmRunxb Type 1 and Type 3 isoforms are largely similar, except for the absence of exon 5 in the Type 3, as a result of exon skipping. CmRunxb Type 1 and Type 2 isoforms differ in their C-terminal ends, resulting from the use of alternative termination codon and splice donor/acceptor sites in exons 5 and 6 (Fig. 9A). At the protein level, CmRunxβ and human RUNXβ show high conservation of amino acid residues 1-165 (Fig. 9B), of which the N-terminal 135 amino acids required for its heterodimerization with the α-subunit and DNA binding [67] are almost perfectly conserved. We investigated the expression patterns of Runxb isoforms in various tissues of adult elephant shark by quantitative RT-PCR. All isoforms display a ubiquitous pattern of expression, with high levels of expression in the gill, heart, ovary and testis of the elephant shark (Fig. 9C). The functional significance of these isoforms that are conserved in human, elephant shark and other jawed vertebrates remains to be investigated. In an attempt to identify conserved cis-regulatory elements for Runxb locus, we searched for CNEs in the human and elephant shark Runxb loci but found none (data not shown).


Runx family genes in a cartilaginous fish, the elephant shark (Callorhinchus milii).

Nah GS, Lim ZW, Tay BH, Osato M, Venkatesh B - PLoS ONE (2014)

Exon-intron organization and protein sequence encoded by the elephant shark Runxβ.(A) Schematic representation of the genomic structure and the three transcripts cloned (CmRunxb types 1,2 and 3). Exons are indicated by boxes. The vertical dashed lines indicate internal splice sites located within a coding exon. The 5′ and 3′UTRs are represented as crosshatched boxes. (B) Alignment of elephant shark and human RUNXβ amino acid sequences using ClustalW. Conserved residues are shaded grey. Cm, Callorhinchus milii; Hs, Homo sapiens. (C) Expression patterns of elephant shark Runxb transcripts. Relative expression levels of Runxb Types 1+2 and Type 3 in various tissues of the elephant shark determined by qRT-PCR.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974841&req=5

pone-0093816-g009: Exon-intron organization and protein sequence encoded by the elephant shark Runxβ.(A) Schematic representation of the genomic structure and the three transcripts cloned (CmRunxb types 1,2 and 3). Exons are indicated by boxes. The vertical dashed lines indicate internal splice sites located within a coding exon. The 5′ and 3′UTRs are represented as crosshatched boxes. (B) Alignment of elephant shark and human RUNXβ amino acid sequences using ClustalW. Conserved residues are shaded grey. Cm, Callorhinchus milii; Hs, Homo sapiens. (C) Expression patterns of elephant shark Runxb transcripts. Relative expression levels of Runxb Types 1+2 and Type 3 in various tissues of the elephant shark determined by qRT-PCR.
Mentions: We cloned the full-length coding sequence of the elephant shark gene encoding the β-subunit of the CBF heterodimeric complex, Runxb. The exon-intron organization of elephant shark Runxb (CmRunxb) gene is identical to that of its human ortholog (Fig. 9A) [66]. Furthermore, CmRunxb is transcribed into at least three isoforms that are homologous to the human RUNXB type 1, 2 and 3 isoforms. The CmRunxb type 1, 2 and 3 isoforms encode proteins of 188, 185 and 156 amino acids, respectively (Fig. 9B). CmRunxb Type 1 and Type 3 isoforms are largely similar, except for the absence of exon 5 in the Type 3, as a result of exon skipping. CmRunxb Type 1 and Type 2 isoforms differ in their C-terminal ends, resulting from the use of alternative termination codon and splice donor/acceptor sites in exons 5 and 6 (Fig. 9A). At the protein level, CmRunxβ and human RUNXβ show high conservation of amino acid residues 1-165 (Fig. 9B), of which the N-terminal 135 amino acids required for its heterodimerization with the α-subunit and DNA binding [67] are almost perfectly conserved. We investigated the expression patterns of Runxb isoforms in various tissues of adult elephant shark by quantitative RT-PCR. All isoforms display a ubiquitous pattern of expression, with high levels of expression in the gill, heart, ovary and testis of the elephant shark (Fig. 9C). The functional significance of these isoforms that are conserved in human, elephant shark and other jawed vertebrates remains to be investigated. In an attempt to identify conserved cis-regulatory elements for Runxb locus, we searched for CNEs in the human and elephant shark Runxb loci but found none (data not shown).

Bottom Line: Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3.Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci.Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The Runx family genes encode transcription factors that play key roles in hematopoiesis, skeletogenesis and neurogenesis and are often implicated in diseases. We describe here the cloning and characterization of Runx1, Runx2, Runx3 and Runxb genes in the elephant shark (Callorhinchus milii), a member of Chondrichthyes, the oldest living group of jawed vertebrates. Through the use of alternative promoters and/or alternative splicing, each of the elephant shark Runx genes expresses multiple isoforms similar to their orthologs in human and other bony vertebrates. The expression profiles of elephant shark Runx genes are similar to those of mammalian Runx genes. The syntenic blocks of genes at the elephant shark Runx gene loci are highly conserved in human, but represented by shorter conserved blocks in zebrafish indicating a higher degree of rearrangements in this teleost fish. Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3. Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci. Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu. In summary, our analysis reveals that the genomic organization and expression profiles of Runx genes were already complex in the common ancestor of jawed vertebrates.

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