Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice

Abstract

The stage- and tissue-specific expression of many eukaryotic genes is regulated by cis-regulatory elements, some of which are located in proximity to the start site of transcription whereas others have been identified at considerable distances. In previous studies we have identified far upstream DNase I-hypersensitive sites in the murine alpha1(I) collagen (Col1a1) gene, which may play a role in the regulation of this abundantly expressed gene. Here we have cloned several of these sites into reporter gene constructs containing the Col1a1 promoter driving the green fluorescent protein (GFP) reporter gene and tested their possible functions in transfection experiments and transgenic mice. In transient and stable transfections none of the hypersensitive sites had a significant effect on Col1a1 promoter activity, indicating that they do not contain a classical transcriptional enhancer. In transgenic animals one element located at -18 to -19.5 kb enhanced the position-independent activity of the linked Col1a1 promoter and may be part of a locus control region. Another element located at -7 to -8 kb specifically enhanced reporter gene expression in the uteri of transgenic mice, suggesting that it contains a novel transcriptional enhancer that may be involved in the regulation of type I collagen expression in tissue remodeling in the uterus during the estrous cycle. Our studies also demonstrate the versatility of the GFP reporter gene for use in transgenic animals because it can be analyzed in live animals, whole mount embryos, histological thin sections, or primary cell cultures, and it can be quantified very sensitively in tissue or cell extracts using a fluorometer.

FIG. 1

Reporter gene constructs. The vertical arrows in the uppermost line show the location of previously mapped DNase I-hypersensitive sites 3–9 in the 5′-flanking region of the murine Col1a1 gene (6,30). The vertical bar shows part of the first exon and the horizontal arrow the start of transcription. Below the reporter gene constructs used in this study are shown schematically. pCol9GFP contains 3.2 kb of the Col1a1 promoter linked to the EGF reporter gene. The other constructs contain the various hypersensitive sites as indicated.

FIG. 2

Upstream hypersensitive sites show marginal effect on Col1a1 promoter activity in stable transfections. The indicated reporter constructs were stably transfected into NIH 3T43 fibroblasts and GFP expression analyzed by FACS and compared to untransfected cells as described in Materials and Methods.

FIG. 3

Upstream hypersensitive site HS8,9 significantly enhanced position-independent transgene expression. Transgene-containing animals were identified by PCR amplification of GFP sequences in tail DNA and transgene-expressing animals by fluorescence analysis in tail and other tissues as described in the text.

FIG. 4

GFP expression in a whole mount embryo containing the reporter gene pCol9GFP-HS4,5 and a negative littermate at day 18 of embryonic development.

FIG. 5

GFP expression in whole mount embryos containing the different reporter genes (see the text) at various stages of embryonic development. (A) day 12, (B) day 14, (C) day 16, (D) day 17, (E) day 18 (p.c).

FIG. 6

HS4,5 enhanced GFP expression in uterus of transgenic mice. Extracts were prepared from tails and uteri of transgenic animals and GFP expression determined by fluorometry as described in Materials and Methods. The data are derived from comparing GFP expression in age-matched offspring from all six founders containing construct pCol9GFP with offspring from seven founders containing pCol9GFP-HS4,5 and offspring from all seven founders containing pCol9GFP-HS8,9 and are shown as expression in uterus in percent of expression in tail of the same animal. The enhanced uterus-specific expression in animals containing HS4,5 is statistically highly significant (p < 0.001).

FIG. 7

GFP expression in the uterus of a transgenic animal. Thin sections of the uterus of an offspring of founder 2033 containing the construct pCol9GFP-HS4,5 were prepared as described in Materials and Methods and analyzed under the fluorescent microscope with filters specific for DAPI stain (A) or GFP (B). Strong fluorescence can be seen in the endometrium (E) and myometrium (M), but not in the epithelial cells lining the uterine lumen (L) or uterine glands (G).

FIG. 8

Correlation between relative GFP expression in the tails of transgenic animals and percentage of GFP-expressing cells in primary dermal fibroblast cultures. Primary dermal fibroblast cultures were established from each of the founders as described in Materials and Methods and the number of expressing cells determined by microscopy and plotted against the relative level of GFP expression in the tails of each animal.