Limits...
A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue.

Zhao H, Innes J, Brooks DC, Reierstad S, Yilmaz MB, Lin Z, Bulun SE - Reprod. Biol. Endocrinol. (2009)

Bottom Line: Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA.Dexamethasone significantly induced activity of this adipose-specific promoter region.These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. h-zhao@northwestern.edu

ABSTRACT

Background: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters.

Methods: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads.

Results: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone.

Conclusion: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.

Show MeSH

Related in: MedlinePlus

Expression of adipose-specific transcripts of the Cyp19a1 gene in different mouse tissues using RT-PCR (A) and in gonadal fat using real-time RT-PCR(B). Tissue-specific primer pairs were used. Each forward primer was located in the tissue-specific first exon. Reverse primers were located in coding exon II. Tad: transcript in adipose tissue, Tgon: transcript in gonad, Ttes: transcript in testis, Tbr: transcript in brain, M: DNA ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2684739&req=5

Figure 4: Expression of adipose-specific transcripts of the Cyp19a1 gene in different mouse tissues using RT-PCR (A) and in gonadal fat using real-time RT-PCR(B). Tissue-specific primer pairs were used. Each forward primer was located in the tissue-specific first exon. Reverse primers were located in coding exon II. Tad: transcript in adipose tissue, Tgon: transcript in gonad, Ttes: transcript in testis, Tbr: transcript in brain, M: DNA ladder.

Mentions: To determine the distribution of Tad, Tov, Ttes, and Tbr in male gonadal fat, ovary, testes, and hypothalamus, exon-specific RT-PCR analysis of RNAs was performed using 5'-UTR-specific forward primers and reverse primers located in coding exon II. Tad and Tov were expressed in male gonadal fat (Figure 4A). Those results were confirmed by real-time RT-PCR in 10-week-old and 16-week-old mouse gonadal fat (Figure 4B). Tbr and Tov were found in the hypothalamus. Tov was the only Cyp19a1 transcript expressed in the ovary. All 4 transcripts were found in the testis, though Tov was the major transcript. Thus, we suggested renaming the previously described Tov as gonadal-specific transcript (Tgon).


A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue.

Zhao H, Innes J, Brooks DC, Reierstad S, Yilmaz MB, Lin Z, Bulun SE - Reprod. Biol. Endocrinol. (2009)

Expression of adipose-specific transcripts of the Cyp19a1 gene in different mouse tissues using RT-PCR (A) and in gonadal fat using real-time RT-PCR(B). Tissue-specific primer pairs were used. Each forward primer was located in the tissue-specific first exon. Reverse primers were located in coding exon II. Tad: transcript in adipose tissue, Tgon: transcript in gonad, Ttes: transcript in testis, Tbr: transcript in brain, M: DNA ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2684739&req=5

Figure 4: Expression of adipose-specific transcripts of the Cyp19a1 gene in different mouse tissues using RT-PCR (A) and in gonadal fat using real-time RT-PCR(B). Tissue-specific primer pairs were used. Each forward primer was located in the tissue-specific first exon. Reverse primers were located in coding exon II. Tad: transcript in adipose tissue, Tgon: transcript in gonad, Ttes: transcript in testis, Tbr: transcript in brain, M: DNA ladder.
Mentions: To determine the distribution of Tad, Tov, Ttes, and Tbr in male gonadal fat, ovary, testes, and hypothalamus, exon-specific RT-PCR analysis of RNAs was performed using 5'-UTR-specific forward primers and reverse primers located in coding exon II. Tad and Tov were expressed in male gonadal fat (Figure 4A). Those results were confirmed by real-time RT-PCR in 10-week-old and 16-week-old mouse gonadal fat (Figure 4B). Tbr and Tov were found in the hypothalamus. Tov was the only Cyp19a1 transcript expressed in the ovary. All 4 transcripts were found in the testis, though Tov was the major transcript. Thus, we suggested renaming the previously described Tov as gonadal-specific transcript (Tgon).

Bottom Line: Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA.Dexamethasone significantly induced activity of this adipose-specific promoter region.These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. h-zhao@northwestern.edu

ABSTRACT

Background: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters.

Methods: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads.

Results: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone.

Conclusion: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.

Show MeSH
Related in: MedlinePlus