B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position ؊139 to ؊146 from the cap site) and fp2 (at ؊157 to ؊164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.Mouse mammary tumor virus (MMTV) is a type B retrovirus which causes carcinomas of the mammary gland in females of susceptible mouse strains (8, 13). The carcinogenic transformation results from transcriptional activation of a particular class of cellular oncogenes (int genes; reviewed in reference 59). The mammary gland specificity of the oncogenic property of MMTV depends on the high viral replication rate and consequent high reinfection rate in the mammary epithelial cells, which are stimulated by pregnancy-related hormones. This hormonal stimulation has made the MMTV promoter the beststudied model for investigating the regulation of gene expression by steroid hormones (for a review, see references 7 and 51). Glucocorticoids, progesterone, and androgens (but not estrogens) strongly stimulate the rate of MMTV transcription through the binding of hormone-receptor complexes in the hormone regulatory element (HRE) (see Fig. 1) region of the proviral DNA, located upstream of the promoter in the long terminal repeat (LTR). Binding sites for additional transcription factors have been characterized, in particular for CTF (also called nuclear factor 1 [NF-1]) and Oct-1 in the HRE, and for mammary gland-specific factors further upstream in the LTR (reviewed in reference 31).During primary infection, MMTV is transmitted in the milk from the mother to the newborn and is taken up in the intestine, where it infects local lymphocytes (rev...

Tissue-specific transcription is advantageously investigated by using viral promoters, which are selected for compact regulatory elements. Mouse mammary tumor virus (MMTV) has adapted to specialized cell types and targets initially B lymphocytes. We previously showed that, in B-cell lines, glucocorticoid-induced MMTV transcription requires an ETS family factor, GA-binding protein (GABP), bound in tandem to the MMTV DNA next to the glucocorticoid receptor (GR). We now report that transforming growth factor ␤ (TGF-␤) superinduces this response up to 10-fold through binding of its effectors, Smads, between the GABP-binding motifs. The basal level was unaffected. The TGF-␤-glucocorticoid cooperation also depended on GR and GABP binding, was transferable to another promoter, and occurred both with transiently transfected and with integrated templates. Smad3 associated in vitro with GR, with GABP␣ (via the MH2 domain), and with GABP␤, Smad4 only with GABP␣. Interactions of Smad3 with GABP (when coexpressed or endogenous to B cells) were shown by coprecipitation and by mammalian two-hybrid assay. This composite DNA element integrates three signaling pathways deriving from TGF-␤, glucocorticoid hormones, and a unique ETS factor, and may allow MMTV to exploit factors from the milk. It may as well indicate novel possibilities for cellular regulatory networks.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position ؊139 to ؊146 from the cap site) and fp2 (at ؊157 to ؊164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.