January 1, 2012;
A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue.
The thermoregulatory function of brown adipose tissue
(BAT) is due to the tissue
-specific expression of uncoupling protein 1 (UCP1
) which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1
transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulfite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1
expression between white and BAT. Tissue
-specific expression of UCP1
has been proposed to be regulated by a conserved 5'' distal
enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in five eutherians as well as marsupials, monotremes, amphibians, and fish, is present in pigs despite UCP1
having become a pseudogene, and that absence of the enhancer element does not relate to BAT-specific UCP1
expression. We identify an additional putative 5'' regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1
expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1
5'' untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1
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Figure 1. Expression and UCP1 promoter percentage CpG methylation. (A) bovine UCP1 mRNA expression by qRTPCR. Methylation sensitive restriction digest determination of (B) bovine, (C) human CpG islands, (D) human enhancer, and (E) bisulfite mapping determination of the percentage methylation of 12 CpGs within the bovine CpG island, in adipose tissues and liver. UCP1 mRNA (A) is expressed relative to ribosomal 18S mRNA. The data are presented as a percentage methylation compared to each respective mock methylated sample for the (B) bovine 288 bp (□) and the 407 bp (■) products and (C) human 173 bp (□) and the 426 bp (■) products and (D) human enhancer (see Materials and Methods). The amount of UCP1 promoter DNA was quantified by qPCR relative to ribosomal 18S DNA. (E) CpG dinucleotide methylation in the Ucp1 proximal promoter in newborn bovine brown (□) and subcutaneous white adipose tissue (■), and liver (). For comparison, values for the mouse enhancer (ENH) BAT, WAT, and liver are presented. DNA was extracted, bisulfite modified, amplified by PCR, and pyrosequenced to determine CpG methylation over positions 1–12 of the Ucp1 promoter (see Materials and Methods). Missing liver values are due to failed analyses. Values are means ± SEM from at least three replicates except for (D) which represents the average of duplicates ± SD *** BAT significantly greater than other tissues (p <0.001).
Figure 2. Map of the relative positions of the conserved enhancer, putative regulatory region and predicted CpG island in the UCP1 promoter of 29 species. All genes are shown in 5′–3′ orientation. Arrow represent the region of the UCP1 coding sequence. Differences in arrow length are likely to reflect relative differences in intron sizes.
Figure A1. Gene synteny around the UCP1 locus. Relative positions of UCP1 orthologs in four representative species showing conservation of synteny. Positions of conserved upstream regions and predicted CpG islands are shown. Gaps in current genome build are shown as hashed boxes. For details see Table A2.
Figure A2. Evolutionary conservation of the regions of the UCP1 5′ UTR enhancer and putative regulatory region. Pairwise comparison of cow-mouse (A) and cow-human (B) genomic DNA 5 Kb upstream of UCP1 gene rVISTA (Loots et al., 2002) using the AVID alignment algorithm (Bray et al., 2003). Conserved regions (>70% conserved in 100 bp window are shaded). A highly conserved peak is visible at approximately −3.6 Kb within the conserved enhancer region. A second conserved region approximately −1.1 to −1.6 kb is conserved between cattle and human but is missing in the mouse genome. Within peaks of sequence similarity are a number of conserved transcription factor binding sites of interest (CEBP, CREB, DR1, DR3, DR4, PPAR) marked with the bars.
Figure A3. Partial alignment of conserved enhancer region in 20 vertebrate species, approximately −3800 bp of human UCP1. For genome coordinates and full alignment, see Appendix.
Figure A4. (A) Alignment of conserved enhancer region in human, bovine, rat, and mouse, approximately −3800 bp of human UCP1. Positions of known transcription factor binding sites taken from Jastroch et al. (2008) (B) Partial alignment of conserved putative regulatory region (PRR) approximately −2200 to −2700 bp of human UCP1. For genome coordinates and full alignment, see Appendix.
Figure A5. Sequence of the conserved enhancer region in 20 vertebrate species.
The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets.