A 500-bp region, approximately 40 kb upstream of the human CYP19 (aromatase) gene, mediates placenta-specific expression in transgenic mice

Abstract

In humans, aromatase P450 (product of CYP19 gene), which catalyzes conversion of C19 steroids to estrogens, is expressed in a number of tissues, including ovary, adipose, and syncytiotrophoblast of the placenta. The 5' untranslated regions of CYP19 mRNA transcripts in these tissues are encoded by different tissue-specific first exons, which are spliced onto a common site just upstream of the translation initiation site in exon II. In placenta, the 5' untranslated region of CYP19 mRNA transcripts is encoded by exon I.1, which lies approximately 40 kb upstream of exon II. To map genomic sequences required for placenta-specific CYP19 expression, fusion genes containing 2,400 and 501 bp of placenta-specific exon I.1 5' flanking DNA linked to the human growth hormone gene (hGH), as reporter, were introduced into transgenic mice. Expression of CYP19(I.1):hGH fusion genes containing as little as 501 bp of 5' flanking DNA was placenta-specific and developmentally regulated. Furthermore, transgene expression occurred specifically in the labyrinthine trophoblast of the mouse placenta, which contains syncytial cells that may be analogous to the human syncytiotrophoblast. We show that a relatively small segment of DNA (approximately 500 bp) >40 kb upstream of the protein coding region of a human gene is able to direct expression in an appropriate tissue- and cell-specific manner in transgenic mice. These findings suggest that 5' flanking DNA within 501 bp of exon I.1 of the human CYP19 gene contains cis-acting elements that bind placenta-specific transcription factors that are conserved between humans and mice.

Figure 1

Schematic diagram of human CYP19(I.1):hGH transgenes. The human CYP19 DNA sequences encoding the first 103 bp of exon I.1 are shown as a solid box, whereas the 5′ flanking DNA is depicted as a solid line. The arrow indicates the position of the transcription initiation site and direction of transcription. The DNA encoding the hGH gene is represented by an open box. The positions of putative regulatory elements that we have found to be functionally important (10) are shown. These elements include the GC box (−233 bp), a hexameric (Hex, −183 bp) element, and the TATA box (−27 bp).

Figure 2

Expression of the CYP19(I.1)₋₂₄₀₀:hGH transgene is placenta-specific in two independent E17.5 founders. Total RNA (30 μg) isolated from various tissues of two E17.5 founder mice (A7-6 and A4-6) was analyzed for hGH mRNA by Northern blotting by using a full-length ³²P-labeled hGH cDNA as probe as described in Materials and Methods.

Figure 3

CYP19(I.1)₋₅₀₁:hGH fusion gene is expressed in placenta of transgenic mice. Aliquots of total RNA (30 μg) isolated from placenta of an E17.5 F₁ mouse or from various tissues of an adult F₁ male or female mouse (line 2219, Upper, or line 2218, Lower) carrying the CYP19(I.1)₋₅₀₁:hGH transgene were analyzed by Northern blotting by using a ³²P-labeled hGH cDNA probe.

Figure 4

Expression of a CYP19(I.1)₋₅₀₁:hGH fusion gene is detectable in transgenic mouse placenta as early as E10.5 and is developmentally regulated. Placentae at E10.5, E15.5, and E17.5 were obtained from a single line of transgenic mice carrying the CYP19(I.1)₋₅₀₁:hGH fusion gene. Total RNA was isolated and analyzed by Northern blotting for hGH expression by using a ³²P-labeled hGH cDNA probe. To evaluate loading efficiency, the blot was stripped and reprobed with a ³²P-labeled cyclophilin cDNA.

Figure 5

Expression of a CYP19(I.1)₋₅₀₁:hGH fusion gene is restricted to labyrinthine trophoblast. Placental tissues obtained from E10.5 or E17.5 fetal mice carrying the CYP19(I.1)₋₅₀₁:hGH (A– C) transgene were processed for in situ hybridization by using a ³⁵S-labeled antisense hGH cRNA probe and exposed to photographic emulsion for 1–2 weeks. Bright- and dark-field microscopy then were performed. (A) Dark-field micrograph of placental tissue section from an E10.5 transgenic mouse carrying the CYP19(I.1)₋₅₀₁:hGH fusion gene hybridized with radiolabeled hGH antisense cRNA probe. (B) Bright-field micrograph of the hematoxylin-stained E10.5 placental tissue section shown in A. (C) Dark-field micrograph of placental tissue section from an E17.5 transgenic mouse carrying the CYP19(I.1)₋₅₀₁:hGH fusion gene incubated with radiolabeled hGH antisense cRNA probe. (D) Dark-field micrograph of E17.5 placental section from a nontransgenic mouse incubated with radiolabeled hGH antisense cRNA probe. gc, trophoblast giant cell; sp, spongiotrophoblast; lab, labyrinthine trophoblast; ud, uterine decidual tissue.