The basic leucine zipper containing activating transcription factors (ATFs) modulates the expression of growth-regulating genes. In this study, we sought to determine specifically the consequences of ATF4 expression on mammary gland development in transgenic mice. Overexpression of ATF4 severely impaired normal development of the mammary gland, which was associated with reduced proliferation and differentiation of mammary alveolar epithelium and up-regulation of p21 WAF1 and p27 Kip1 . In addition, there was also impaired lactation accompanied by decreased expression of ␣-lactoalbumin, whey acidic protein, and -casein, possibly because of the down-regulation of STAT5a tyrosine phosphorylation. Mammary gland involution in ATF4-transgenic mice was accelerated, compared with wild type littermates by whole mount analysis. In addition, day 18 of lactation in transgenic mice was phenotypically equivalent to day 3 of involution in wild type mice, as determined by the TUNEL assay and expression of Bax. The concentration of the proapoptotic molecule caspase-3 was increased during lactation in ATF4-transgenic animal. Mammary glands from ATF4-transgenic mice also showed significant nuclear translocation of activated STAT3 and up-regulation of one of its target genes, insulin-like growth factor-binding protein-5, which is thought to facilitate apoptosis by sequestering insulin-like growth factor. Together, these findings suggest that ATF4 may play a role during mammary gland development and that down-regulation of ATF4 may be important for the onset of involution in the mammary gland.Among the main regulatory elements that contribute to transcriptional regulation of extracellular signals are the cAMPresponsive element (CRE) 1 and activation protein (AP-1) sequence motifs. It is increasingly accepted that the CRE site (TGACGTCA) is recognized by a family of basic leucine zippercontaining proteins known as CRE-binding proteins (CREB) or activating transcription factors (ATFs), including ATF4. Because ATF binding sites are present in several growth-regulating gene promoters, ATFs are believed to be involved in different regulatory circuits, allowing cells to integrate signals from distinct pathways. The mammalian ATF4 protein has been demonstrated to form heterodimers with members of the AP-1 and C/EBP family of proteins, including Fos (1), Jun (1-3), JunD (4), and several C/EBP proteins (C/EBP␣, C/EBP/CRP2, C/EBP, and C/EBP⑀/CRP1) (5-8). ATF4 acts both as a transcriptional activator (7, 9 -14) and a transcriptional repressor (4,(15)(16)(17)(18)(19), presumably by sequestering other regulatory factors away from promoters. ATF4 also interacts with the coactivator CREB-binding protein and components of the general transcription machinery, such as the TATA-binding protein, TFIIB and the RAP30 subunit of TFIIF (20). Overexpression of ATF4 in murine NIH3T3 fibroblasts reduces the ability of the ectopically expressed Ras oncogene to transform cells as judged by cellular morphology and foci formation and has thus been proposed to r...