The Homeodomain Protein CDP Regulates Mammary-Specific Gene Transcription and Tumorigenesis

The CCAAT displacement protein (CDP) family constitutes a highly conserved group of homeoproteins that are involved in cell growth, differentiation, and development. CDP (also known as Cut in Drosophila melanogaster, Clox in dogs, and Cux in mice) is a transcriptional repressor that contains four DNA-binding domains, three Cut repeats (CR1, -2, and -3), and a homeodomain (32). CDP is involved in suppressing transcription from many cellular genes, including c-myc, transforming growth factor β type II receptor, phox, CD8, immunoglobulin heavy chain, and T-cell receptor β-chain (3, 7, 12, 19, 22, 46), as well as viral genes, including those from mouse mammary tumor virus (MMTV) and human papillomaviruses (HPVs) (1, 55). In addition, CDP has been implicated as a tumor suppressor gene in human breast carcinomas and uterine leiomyomas (28, 47, 53). Many genes regulated by CDP are expressed in highly differentiated cells. CDP DNA-binding activity is downregulated during B-cell and myeloid cell development (22, 46), and CDP expression in the kidney is inversely related to the degree of cellular differentiation (44). We have shown that CDP is a repressor of MMTV long terminal repeat (LTR) reporter gene expression in cultured cell lines and that CDP-binding activity to the LTR declines during mammary gland development (55). These results suggest that CDP functions as a transcriptional repressor that inhibits expression of differentiation-specific genes in several tissues. The retrovirus MMTV is a paradigm for hormone-regulated gene expression in the mammary gland. Virus is transmitted exogenously from mothers to offspring through virally infected milk (11). Gut-associated B and T lymphocytes are infected by MMTV and amplified by virally encoded superantigen (Sag) prior to transmission of virus to mammary cells during puberty. Hormonal stimulation during pregnancy and lactation increases MMTV transcription and replication, allowing mutagenic integration events and the development of mammary tumors (11). MMTV also is transmitted through the germ line of most inbred mouse strains as endogenous proviruses (8). Because most viruses specify very few genes, they must rely on their host cells for replication. In particular, many retroviruses show cell-type-specific replication and tumorigenesis, and studies of such viruses have elucidated many aspects of diverse cellular processes. Current evidence suggests that tissue-specific MMTV replication is controlled primarily at the transcriptional level using host factors (24). Lymphomagenic MMTV strains invariably have a deletion of 350 to 500 bp of negative regulatory elements (NREs) within the LTR U3 region (18). These deletions remove the binding sites for two homeodomain-containing transcriptional repressors, special AT-rich binding protein 1 (SATB1) and CDP (23, 24). Studies in transgenic mice showed that mutation of a SATB1-binding site within the MMTV LTR elevates viral transcription in lymphoid tissues (24). Two CDP binding sites have been mapped to the promoter-proximal portion of the MMTV NRE, and mutation of either of these binding sites elevated reporter gene expression from the MMTV LTR (55). Other functional CDP binding sites have been mapped to the promoter-distal NRE (54). These data indicate that negative regulation of transcription is a primary method for regulating tissue-specific MMTV expression. Since CDP binding to MMTV NREs is high in virgin mammary glands, we predicted that loss of CDP binding would increase MMTV transcription in early stages of mammary development, thereby altering the kinetics of virally induced tumors. To test whether CDP has a direct role in MMTV-specific transcription and tumorigenesis, we constructed infectious proviruses with different CDP binding site mutations. Mice infected with these mutants showed increased MMTV expression in the virgin mammary gland and accelerated appearance of breast tumors. To determine specific effects by CDP on transcription in the mammary gland, we also analyzed gene expression in two different strains of CDP-mutant mice, lacking either CR1 or the entire C terminus including the homeodomain (42, 43). RNA levels of endogenous MMTV, whey acidic protein (WAP), and α-lactalbumin, but not glyceraldehyde-3-phosphate dehydrogenase (gapdh) or WDNM1, were elevated in virgin mammary glands of both mutant strains compared to wild-type controls. However, only animals with one-half the wild-type CDP levels had increased β-casein expression. These results indicate that different CDP domains contribute to negative regulation of differentiation-specific genes in the mammary gland.