Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility

Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1(tm2Ejn), referred to as Delta C) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Delta C(-/-)) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and Delta C(-/-) MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1(tm2Ejn/tm2Ejn) mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions.     

A composite enhancer element directing tissue-specific expression of mouse mammary tumor virus requires both ubiquitous and tissue-restricted factors

Mouse mammary tumor virus (MMTV) expression is restricted primarily to mammary epithelial cells. Sequences responsible for both the mammary-specific expression of MMTV and the activation of cellular oncogenes are located within two enhancer elements at the 5'-end of the long terminal repeat. Whereas the Ban2 enhancer (-1075 to -978) has been well characterized, the mammary-specific enhancer of MMTV from -956 to -862 has only recently been recognized as a key determinant of mammary-specific oncogene activation by MMTV. The present study identifies and characterizes three binding sites located within this element. Transient transfection of deletion and mutation constructs shows that all three sites contribute to the basal expression of MMTV in mammary cells. One of the binding activities (footprint I) is restricted to mammary cells, whereas the other two sites bind factors found in both mammary and nonmammary cells. The multimerized mammary-specific enhancer of MMTV on its own can enhance a minimal promoter in a mammary-specific fashion. However, the FpI binding site alone cannot mediate this effect. Thus, it is the binding of multiple factors in a combinatorial fashion that mediates the mammary-restricted expression of MMTV.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein

The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.