Differentiation-Induced Cleavage of Cutl1/CDP Generates a Novel Dominant-Negative Isoform That Regulates Mammary Gene Expression

Abstract

Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland.

We thank members of the Dudley laboratory and Jon Huibregtse for helpful comments on the manuscript. Alain Nepveu generously provided the N-terminal CDP-specific antibody. We also are grateful to Mina Bissell and members of her laboratory for providing SCp2 cells and antibody specific for β-casein.

This work was supported by grants R01CA34780 and DAMD17-01-1-0424 from the National Institutes of Health and the U.S. Army Medical Research and Materiel Command.