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.

FIG. 1

Identification of NFI genes expressed during different stages of mammary gland development. (A) RNA was isolated from virgin, pregnancy day 15 (Preg d15), and lactation day 2 (Lact d2) mouse mammary glands, and RT-PCR was performed with degenerate primers Deg1 and Deg2. The 486-bp amplified product was digested with BamHI, BpuI1021, and NarI and then separated on a 2% agarose gel. The bands in each lane (1, 2, and 3) represent the NFI genes in virgin, pregnancy day 15, and lactation day 2 mice, respectively. The arrows indicate the undigested 486-bp NFI-B, 394-bp NFI-X, 327-bp NFI-A, and ≈200-bp NFI-C gene fragments. The numbers on the left side represent the molecular size markers. (B) RT-PCR was performed with degenerate primers and [³²P]dATP, and the BamHI-, BpuI1021-, and NarI-digested 486-bp fragments were separated on a 10% polyacrylamide gel. Lanes 1, 4, and 7 represent reactions using the L19 primers, which generated a 200-bp product, and lanes 2, 5, and 8 represent the degenerate primer-amplified 486-bp band of the conserved N-terminal region of each NFI gene (−E, not digested with enzymes). Lanes 3, 6, and 9 represent the expression of each NFI gene in virgin, pregnancy day 15, and lactation day 2 mammary glands of mice (+E, digested with enzymes after amplification). The arrows indicate the individual NFI genes. The numbers on the right side represent the molecular size markers.

FIG. 2

RNase protection assay for identification of the expression pattern of four NFI genes at different stages of mammary gland development. RNA was isolated from mammary gland of virgin (V), pregnancy day 15 (P), lactation day 2 (L), and involution stage (I) mice and from mouse brain (Br). RNase protection assays were performed using antisense probes for each NFI gene along with a mouse β-actin control. (A) Schematic representation of the genomic structure of three NFI isoforms expressed in the mouse mammary gland during lactation. (B) Lanes 1 to 5 represent the NFI-A, and lanes 6 to 10 represent NFI-B. (C) Lanes 1 to 5 represent NFI-C, and lanes 6 to 10 represent NFI-X. Upper arrows indicate the 486-bp protected band for each NFI gene, and the asterisk indicates the nonspecific bands. The expression of each NFI gene at each developmental stage was analyzed using the PhosphorImager, and the results are graphically represented in panel D. Error bars denote the standard error of the mean (SEM) of three independent determinations. An asterisk above the error bar represents the statistical significance of the data (P < 0.05).

FIG. 3

Cooperativity of GR and STAT5A on the WAP CoRE. (A) Schematic diagram of the pWAPtk-luciferase construct. (B) GR and STAT5A cooperativity. The pWAPtk-luciferase construct (1 μg) was transiently transfected into JEG-3 cells along with GR (0.2 μg), STAT5A (1 μg), PrlR (0.3 μg), and RSV-β-gal (0.3 μg). Twenty-four hours after induction with hormones (1 μg/ml of both HC and Prl), luciferase expression was measured and transfection efficiencies were normalized to β-galactosidase activity. Cells were induced with HC, Prl, and both HC and Prl, as indicated. (C) Cooperativity of STAT5A and NFI isoforms (0.2 μg, 0.5 μg, and 1 μg of A, B, and X, respectively) for the activation of the WAP CoRE. Three NFI isoforms were transfected into JEG-3 cells along with STAT5A, PrlR, pWAPtk-luciferase, and RSV-β-gal, and the cells were induced with Prl. Cells were transfected with STAT5A and PrlR and induced with Prl and with NFI-A, -B, and -X, respectively, as shown. (D) Cooperativity of GR, STAT5A, and NFI isoforms on the WAP CoRE. Cells were transiently transfected with GR, STAT5A, the PrlR, and pWAPtk-luciferase along with RSV-β-gal. In addition to these constructs, cells were transfected with either NFI-A, -B, or -X. Cells were induced with HC, Prl, or both HC and Prl as indicated. Error bars represent the standard error of three independent determinations. (E) Expression of the three NFI isoforms in JEG-3 cells. Total proteins were isolated from the cells transfected with NFI-X1 (lane 1), NFI-B2 (lane 2), and NFI-A4 (lane 3) and from cells transfected with only the pCH vector (lane 4). These extracts were analyzed on sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis (SDS–8% PAGE), and Western blots were performed using an anti-HA antibody. The molecular sizes of the protein markers are shown.

FIG. 4

GR domains required for cooperative interactions. (A) Schematic diagram of wild-type (WT) GR and mutant GR proteins. The C-terminal mutant is designated GR 3-556, the N-terminal mutant is GR X-795, and the DNA-binding domain (DBD) mutant is GR C482S. (B) The levels of expression of GR and the different GR mutants shown in panel D determined by Western blotting. Lane 1, wild-type GR; lane 2, GR 3-556; lane 3, GR X-795; lane 4, GR C482S. Total protein isolated from transfected cells depicted in panel D was separated by SDS–8% PAGE and blotted with mouse monoclonal anti-GR antibody. The numbers on the left indicate the molecular sizes of the protein markers. (C) Cooperativity of GR with NFI isoforms for the activation of the WAP CoRE. GR was cotransfected with three NFI isoforms into JEG-3 cells along with pWAPtk-luciferase and RSV-β-gal, and the cells were induced with HC. Cells were transfected with only GR and with NFI-A, NFI-B, or NFI-X as illustrated. (D) Analysis of GR and GR mutant cooperativity with NFI-B on the WAP CoRE: cells were transfected with wild-type GR or one of the three GR mutants and NFI-B along with pWAPtk-luciferase and RSV-β-gal and induced with HC. Cells were transfected with wild-type GR or with both NFI-B and wild-type GR or GR 3-556 C-terminal, GR X-795 N-terminal, and GR C482S DBD mutants, respectively, as shown. Error bars represent the standard error of three independent determinations.

FIG. 5

Cooperative interactions on the MMTV promoter. (A) Schematic diagram of MMTV-luciferase construct. (B) Cooperativity of STAT and GR on the MMTV promoter. MMTV-Luc was transfected with only GR, with only STAT5A, and with both GR and STAT into JEG-3 cells, and the cells were induced with HC, with Prl, and with both hormones as illustrated. (C) Cooperative activation of GR with NFI isoforms on the MMTV promoter. MMTV-Luc and RSV-β-gal were cotransfected into JEG-3 cells along with GR alone, with both GR and NFI-A, with both GR and NFI-B, and with both GR and NFI-X, as shown. The cells were induced with HC. (D) Cooperativity among GR, STAT5A, and NFI isoforms required for the activation of MMTV. MMTV-Luc was transiently transfected into JEG-3 cells with GR, with STAT5A, with both GR and STAT5A, with GR, STAT5A, and NFI-A, with GR, STAT5A, and NFI-B, and with GR, STAT5A, and NFI-X, as illustrated. The cells were induced with HC, with Prl, and with both hormones, as depicted.

FIG. 6

DNA-binding specificities of different NFI isoforms. (A) DNA-binding specificity of NFI-A to the palindromic motif present on the WAP distal promoter estimated by competition EMSA. The specific DNA-protein complexes (described in Materials and Methods) were made to compete with the same palindromic NFI oligonucleotide (homologous competitor) using increasing concentrations as shown (lanes 2 to 8; no competitor in lane 1). (B) Comparison of DNA-binding specificity of the three NFI isoforms to the WAP palindromic motif. The DNA-binding specificities of NFI-B and -X were also determined along with NFI-A (A) by competition EMSA, and the DNA-protein complex and free probe were quantitated by PhosphorImager. Binding specificities were determined by plotting the percentage of bound/unbound versus competitor added. Open squares represent NFI-A, squares filled with the + sign represent NFI-B, and dotted rectangles represent NFI-X. (C) DNA-binding specificity of NFI-A isoform to the palindromic motif on the adenovirus origin of replication region estimated by competition EMSA. The specific DNA-protein complexes (described in Materials and Methods) were made to compete with the same palindromic NFI oligonucleotide (homologous competitor) using increasing concentrations as shown (lanes 2 to 8; no competitor in lane 1). (D) Comparison of DNA-binding specificity of the three NFI isoforms to the adenovirus palindromic motif. The DNA-binding specificities of NFI-B and -X were also determined along with NFI-A (C) by competition EMSA, and the DNA-protein complex and free probe were quantitated by PhosphorImager. Binding specificities were determined by plotting the percentage of bound/unbound versus competitor added. Open squares represent NFI-A, open circles represent NFI-B, and the open rectangles represent NFI-X.