Differential Interactions of Specific Nuclear Factor I Isoforms with the Glucocorticoid Receptor and STAT5 in the Cooperative Regulation of WAP Gene Transcription

Abstract

The distal region (؊830 to ؊720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice. This study reports the characterization of NFI gene expression during mammary gland development and the identification and cloning of specific NFI isoforms (NFI-A4, NFI-B2, and NFI-X1) from the mouse mammary gland during lactation. Some but not all of these NFI isoforms synergistically activate WAP gene transcription in cooperation with GR and STAT5, as determined using transient cotransfection assays in JEG-3 cells. On both the WAP CoRE and the mouse mammary tumor virus long terminal repeat promoter, the NFI-B isoform preferentially activated gene transcription in cooperation with STAT5A and GR. In contrast, the NFI-A isoform suppressed GR and STAT cooperativity at the WAP CoRE. Finally, unlike their interaction with the NFI consensus binding site in the adenovirus promoter, the DNA-binding specificities of the three NFI isoforms to the palindromic NFI site in the WAP CoRE were not identical, which may partially explain the failure of the NFI-A isoform to cooperate with GR and STAT5A.

The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

“…pRSV-β-gal was used to monitor transfection efficiency and cells were induced with hydrocortisone (HC) and prolactin (Prl). The cooperativity of GR and STAT5A with NFI was only observed with NFI-B2 (7.5 fold, Fig1C), but not with NFI-A4 in confirmation of previous results [2]. However, interestingly the chimeric protein N-AC-B (N-ter of A4 and C-ter of B2, Fig.1C) resulted in a 9-fold induction of luciferase activity with GR and STAT5 as compare to the uninduced control.…”

“…pWAPtk-luciferase, STAT5A, GR, prolactin receptor (PrlR) and the NFI constructs have been described previously [2]. To make the chimeric constructs (pCHNFI N-terA-C-terB and pCHNFI N-terB-CterA) between the NFI-A4 and NFI-B2, the pCHNFI-A4 and B2 were digested with EcoNI and NotI and the N-terminal fragment of A ligated with C-terminal fragment of B and vise versa.…”

“…The double-stranded oligonucleotide was end labeled with [γ -32 P] dATP using polynucleotide kinase (Gibco-BRL), and the probe was purified using p-6 Micro Bio-spin columns (Bio-Rad) followed by trichloroacetic acid precipitation to quantify the amount of labeled probe. The chimeric proteins (N-terminal of NFI-A4 fused with C-terminal of NFI-B2, N-terminal of NFI-B2 fused with Cterminal of NFI-A4) were expressed in JEG-3 cells, and nuclear extracts, isolated as described previously (Mukhopadhyay et al, 2001), were used for the DNA-binding assays. The amount of nuclear extract required for 50 % binding of the labeled probe was calculated, and then for each protein that amount was used for the competition assays.…”

“…Studies from several different laboratories have suggested that the spatially and temporally restricted milk protein gene expression patterns result from combinatorial interactions at the protein-protein and protein-DNA level of members of several families of commonly expressed transcription factors [2].…”

“…A distal region of the rat WAP gene, containing a composite response element (CoRE), which confers mammary gland-specific and hormonally-regulated expression, was identified previously in our laboratory [3,4]. In addition, the function of this CoRE was determined by the cooperative interactions among three transcription factors; a specific NFI isoform, the glucocorticoid receptor (GR) and STAT5A [2].…”