Duplicated Spot 14 genes in the chicken: characterization and identification of polymorphisms associated with abdominal fat traits
Introduction
The Spot 14 gene, also referred to as thyroid hormone responsive Spot 14 (THRSP), encodes a small acidic protein that was discovered in earlier studies of thyroid hormone action in hepatocytes Seelig et al., 1981, Jump et al., 1984, Liaw and Towle, 1984. Although the exact molecular mechanism is not clear, THRSP is implicated as a transcription factor involved in control of lipogenic enzymes. For instance, THRSP is only expressed in lipogenic tissue such as liver, fat and the mammary gland Liaw and Towle, 1984, Jump and Oppenheimer, 1985. THRSP mRNA levels are greatly increased by carbohydrate feeding or insulin-injection and decreased by high plasma glucagon levels or by feeding a diet rich in polyunsaturated fatty acids (Jump et al., 1993). Hepatocytes transfected with a THRSP antisense oligonucleotide express decreased mRNA levels in enzymes involved in the lipogenic pathway [i.e., ATP-citrate lyase (ACLY), fatty acid synthase (FAS) and malic enzyme (ME)] Kinlaw et al., 1995, Brown et al., 1997. Although an increase in lipogenesis was observed in the THRSP knockout mouse, this contradiction could be due to incomplete gene deletion or overcompensation by alternative pathways (Zhu et al., 2001). Homodimers of THRSP interact with and activate chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) in promoting transcription of L-type pyruvate kinase (L-PK) through its interaction with specificity protein 1 (Sp1) binding site (Compe et al., 2001). Furthermore, the THRSP promoter region contains three thyroid response elements (TREs) that work synergistically and interact with far upstream region (FUR) elements to maximize triiodothyronine (T3) responses in hepatocytes (Liu and Towle, 1994). Apparently, the human THRSP promoter responds more robustly to T3 than glucose, while the rat THRSP promoter region is more responsive to glucose than T3 (Campbell et al., 2003).
Chicken Spot 14 (THRSP) was first identified by microarray analysis as a differentially regulated EST (pat.pk0032.c9.f) in livers of chickens divergently selected for fast or slow growth rate Cogburn et al., 2000, Cogburn et al., 2003b. A related EST was revealed by differential mRNA display in liver of genetically fat and lean chickens and subsequently mapped to 1q41–44 (Carre et al., 2001). This chromosomal region in chickens also harbors quantitative trait loci (QTL) for skin fatness (Ikeobi et al., 2002) and abdominal fatness (Lagarrigue et al., 2003). Furthermore, expression of THRSP mRNA is regulated by thyroid hormone status in broiler chickens (Wang et al., 2002).
In this paper, we describe the identification, characterization and expression of duplicated, polymorphic Spot 14 (THRSPα and THRSPβ) genes in the chicken. We also report an association of the THRSPα locus with abdominal fat traits in a Leghorn×broiler resource population.
Section snippets
Chicken EST assembly and DNA sequence analyses
The in silico cDNA sequence of THRSP was assembled from chicken EST sequences generated from two international chicken EST projects Boardman et al., 2002, Cogburn et al., 2004 and those found in public databases (GenBank). Contigs were assembled using CAP3 (Huang and Madan, 1999) with 40-bp overlap and 90% identity; the CAP3 assemblies and a chicken gene index are available on our website (http://udgenome.ags.udel.edu/∼cogburn). Contig and unassembled singlet sequences were used in BlastN and
Identification of THRSPα and THRSPβ paralogs
An in silico cDNA sequence representing chicken THRSPα (Fig. 1A) was assembled from a total of 61 ESTs (UD CAP3 Contig_8452.1) found in the UD chicken EST (http://www.chickest.udel.edu/), the British Biotechnology and Biological Sciences Research Council (BBSRC) chick EST (http://www.chick.umist.ac.uk/) (Boardman et al., 2002) and GenBank databases. The THRSPα contig sequence is 874 bp and it includes two closely located poly(A) signals in the 3′-UTR. No additional sequence was found at the
Discussion
In this paper, we have described the discovery, characterization and expression of duplicated, but distinct, Spot 14 (THRSPα and THRSPβ) genes and VNTR polymorphisms in THRSPα and THRSPβ. The THRSPα locus is associated with abdominal fat traits in a broiler×Leghorn resource population. The phylogenetic analysis of the THRSP protein family among mammals, birds and fishes shows that the chicken THRSP paralogs belong to the THRSP ortholog group, while zebrafish G12 and TC194742 belong to the
Conclusions
We have discovered duplicated paralogs of Spot 14 in the chicken, THRSPα and THRSPβ. The duplicated THRSP genes were identified by sequence analysis of contigs assembled from our chicken EST collection and those in public databases. The transcription of THRSPα and THRSPβ mRNA in liver is controlled by developmental, hormonal and nutritional factors. A computational analysis of THRSP proteins has revealed three highly conserved domains in two structurally related proteins from the THRSP family
Acknowledgements
This work was supported by a competitive USDA grant (USDA-IFAFS Animal Genome Program, Award Number 00-52100-9614) to LAC and by Hatch and State of Iowa funds to SJL. The authors are grateful to Dr. Michel Duclos at INRA, Nouzilly, France, who provided liver RNA samples from fasting and re-feed broiler chickens.
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