Nuclear Factor I and Mammary Gland Factor (STAT5) Play a Critical Role in Regulating Rat Whey Acidic Protein Gene Expression in Transgenic Mice

Abstract

The rat whey acidic protein (WAP) gene contains a mammary gland-specific and hormonally regulated DNase I-hypersensitive site 830 to 720 bp 5 to the site of transcription initiation. We have reported previously that nuclear factor I (NFI) binding at a palindromic site and binding at a half-site are the major DNA-protein interactions detected within this tissue-specific nuclease-hypersensitive region. We now show that point mutations introduced into these NFI-binding sites dramatically affect WAP gene expression in transgenic mice. Transgene expression was totally abrogated when the palindromic NFI site or both binding sites were mutated, suggesting that NFI is a key regulator of WAP gene expression. In addition, a recognition site for mammary gland factor (STAT5), which mediates prolactin induction of milk protein gene expression, was also identified immediately proximal to the NFI-binding sites. Mutation of this site reduced transgene expression by approximately 90% per gene copy, but did not alter tissue specificity. These results suggest that regulation of WAP gene expression is determined by the cooperative interactions among several enhancers that constitute a composite response element.

A MGF/STAT5 binding site is necessary in the distal enhancer for high prolactin induction of transfected rabbit αs1‐casein‐CAT gene transcription

“…A distal MGF/STAT5-binding site has already been identified in the rat WAP gene [24]. According to the authors, the presence of this distal binding site is necessary to support a high expression of the gene in transgenic animals.…”

Polymorphisms of beta-lactoglobulin promoter region in three Sicilian goat breeds

“…In most cases, NF-I proteins cooperate with other ubiquitous or tissue-specific TFs whose binding sites are in close proximity. This appears to be the situation for several milk protein genes, including the WAP gene [53]. Binding sites for NF-I have been identified in the ovine promoter [43], using oligonucleotides containing the consensus site of this factor (S2, A1, D2 and A2 elements).…”

“…The effect of these SNPs on binding affinity of TFs, their relationship with BLG gene expression and milk production and composition, and the functional interaction of the TFs with the goat BLG promoter region remain to be determined. a MPBF containing StM (a1), A3S (a2), A1 (a3) consensus site (GGTTCC(N)GGAACC), respectively, as described by Watson et al [42] b NF-I containing S2 (b1), A1 (b2), A2 (b3), D2 (b4) consensus site (TGGC/A(N) 5 GCCAA), respectively, as described by Watson et al [42] c Consensus site for NF-I and MPBF as described by Watson et al [42]; C indicates the SNP -205 C/T found in the Maltese breed and G indicates SNP -197 G/A found in Maltese, Girgentana and Derivata di Siria breeds d Consensus sequence for MAF (gtgGRRGSAAGKgtcc) as described by Mink et al [53] e Consensus sequence for AP-2 identified in TRANSFAC database with the entry R02121; G indicates the SNP -64 G/A found in Girgentana breed…”

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

“…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].…”