A SINGLE NUCLEOTIDE POLYMORPHISM POSSIBLY ASSOCIATED WITH FAT DEPOSITION IS METHYLATED IN THE BOVINE TFAM PROMOTER

1 University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia 2 Corresponding author, e-mail: tanja.kunej@bf.uni-lj.si 3 Washington State University, School of Molecular Biosciences, Pullman, WA 99164-6525, USA, e-mail: zeping_wang@wsu.edu 4 Washington State University, Department of Animal Sciences, Pullman, WA 99164-6351, USA, e-mail: jennifer.michal@wsu.edu 5 Same address as 1, e-mail: peter.dovc@bf.uni-lj.si 6 Same address as 3, e-mail: magnuson@wsu.edu 7 Same address as 4, e-mail: jiangz@wsu.edu A single nucleotide polymorphism possibly associated with fat deposition is methylated in the bovine TFAM promoter Mitochondrial transcription factor A (TFAM) is a nuclear-encoded mitochondrial protein that plays an important role in energy metabolism and is a candidate gene for fat deposition in cattle and human. In the present study, we characterized the methylation status of bovine TFAM promoter flanking the single nucleotide polymorphism (SNP) rs42159487C>T, previously reported to affect fat deposition. Our results showed that the cytosine at this SNP position is methylated and therefore results in gain/loss of the functional CpG locus (5mC>T). Promoter constructs developed based on three TFAM SNPs showed that the C/C/C haplotype associated with fat deposition in beef cattle had lower promoter function/activity than A/T/T haplotype. Our results imply that genetic variability underlying gain/loss of the CpG sites in the nuclear-encoded mitochondrial proteins might be good candidate loci for fat deposition phenotypes.


A single nucleotide polymorphism possibly associated with fat deposition is methylated in the bovine TFAM promoter
Mitochondrial transcription factor A (TFAM) is a nuclear-encoded mitochondrial protein that plays an important role in energy metabolism and is a candidate gene for fat deposition in cattle and human.In the present study, we characterized the methylation status of bovine TFAM promoter flanking the single nucleotide polymorphism (SNP) rs42159487C>T, previously reported to affect fat deposition.Our results showed that the cytosine at this SNP position is methylated and therefore results in gain/loss of the functional CpG locus (5 m C>T).Promoter constructs developed based on three TFAM SNPs showed that the C/C/C haplotype associated with fat deposition in beef cattle had lower promoter function/activity than A/T/T haplotype.Our results imply that genetic variability underlying gain/loss of the CpG sites in the nuclear-encoded mitochondrial proteins might be good candidate loci for fat deposition phenotypes.

INTRODUCTION
Approximately 50 % of gene transcripts encoding mitochondrial proteins decrease with the onset of obesity (Wilson-Fritch et al. 2003).One of these genes, mitochondrial transcription factor A (TFAM) is a strong candidate gene related to fat deposition in mammals (Jiang et al. 2005).In human medicine, TFAM was proposed to be one of the DNA markers and therapeutic targets for predisposition to obesity (Hudson, Lehnert, and Harper 2008) and is also included in the Obesity gene atlas in mammals (Kunej et al. 2013).Our previ-T.KUNEJ et al.
ous study showed significant associations of two closely linked TFAM promoter polymorphisms rs42159489A>C and rs42159488C>T with carcass, meat quality and fat deposition/composition in Wagyu x Limousin F 2 crosses (Jiang et al. 2005).Additionally, a third mutation rs42159487C>T transition was also detected in the bovine TFAM promoter region, which showed significant association with beef marbling score (BMS), subcutaneous fat depth (SFD), and polyunsaturated fatty acids (PUFA) (Jiang et al., 2006).Since the third TFAM gene polymorphism rs42159487C>T resides within a CG dinucleotide of the promoter region we aimed this study to determine the methylation status of the CG dinucleotide to discover if gain/loss of the DNA methylation could be a causative factor associated with fat deposition traits in cattle.Moreover, promoter activities of four constructs developed on haplotype information for these three TFAM promoter SNPs were also analyzed.
where y ijk = ratio of the i-th haplotype in the k-th replication of the j-th experiment, h i = fixed effect of the i-th haplotype, p j = random effect of the j-th experiment, and e ijk = residual.The overall mean is not included in the model, in order to ensure all unknown parameters in the model could be identified (i.e.all parameters are estimated uniquely).Technical details on the assembling of the promoter constructs are described as reported previously (Jiang et al., 2007;Kunej et al., 2007).

RESULTS AND DISCUSSION
We analyzed methylation status of ~ 250 bp of the cattle distal TFAM promoter (nucleotides approximately 28:46080000-28:46080243) flanking the cytosine residing at the polymorphism rs42159487C>T (previously named AAFC03078788.1g.11650C>T) (Figure 1).The DNA extracted from the muscle and recovered after the bisulfite modification was amplified by PCR and sequenced directly.The methylated cytosines remained intact while the unmethylated cytosines were completely converted into uracil following bisulfite treatment and detected as thymine following PCR (Figure 2A).The analysis showed that at rs42159487C>T the cytosine is methylated and that genetic variation C>T causes gain/ loss of the CpG locus.Sequences for the rs42159487 genotype CC, genotype TT and bisulfite treated DNA are shown in Figure 2B.We analyzed bisulfite treated muscle DNA from six animals with rs42159487 C/C and six animals with C/T genotype; in all cases the bisulfite sequencing showed methylation of the cytosine.Additionally, we determined the methylation status for six adjacent CpGs; three of them had methylated and three hemimethylated DNA status (Figure 2A).
Four promoter constructs were developed based on haplotypes among three cattle TFAM promoter SNPs: A/T/C, C/C/C, C/C/T, and A/T/T.As shown in Figure 3, these constructs had different promoter activity patterns in two cell lines: H1299 and HEK293.H1299 cell line was selected as a highly proliferative cell line, which is widely used in research due to its high rate of transgene expression, whereas HEK293 cell line was selected because of its propensity for transfection.In the Wagyu × Limousin F 2 population, we found that C/C/C and A/T/T are the two major haplotypes (Jiang et al., 2009).In general, the former haplotype had lower promoter activity compared to the latter haplotype and the difference among cell lines was more pronounced in the A/T/T haplotype.However, the former haplotype is associated with high marbling score and SFD, while the latter haplotype reduces the expression of both traits.In addition, the substitution rs42159487C>T causes gain/loss of a CpG site.When the aforementioned CpG site is present, it is methylated.These results indicate that reduced promoter function/ activity of the bovine TFAM might be associated with more fat in beef cattle.Therefore, change of TFAM gene expression is consistent with beef marbling or intramuscular fat accumulation.The methylation status of this locus should also be tested in other cattle breeds, tissues and developmental stages.Additional functional studies should be performed, for instance estimating the mitochondrial content through mitochondrial copy number.