Homeoproteins CDP and SATB1 Interact: Potential for Tissue-Specific Regulation

Abstract

Homeoproteins are known to participate in development and cell type specification. The homeoproteins CCAAT displacement protein (CDP) and special AT-rich sequence binding protein 1 (SATB1) have been shown to bind to nuclear matrix-associated regions and to act as repressors of many cellular genes. Moreover, binding of SATB1 to the mouse mammary tumor virus (MMTV) promoter region dramatically affects the tissue-specific transcription of this retrovirus. Because protein-protein interactions are a common means of regulating homeoprotein function, we tested whether SATB1 and CDP interact in vivo and in vitro. SATB1 interacted with CDP through its DNA-binding domain, as demonstrated by glutathione S-transferase (GST) pull-down assays. GST pull-down assays also showed that CDP associated with SATB1 through three of its four DNA-binding domains (CR1, CR2, and the homeodomain). SATB1-specific antisera, but not preimmune sera, precipitated CDP from nuclear extracts, and CDP-specific antisera precipitated SATB1 from the same extracts. FarWestern blotting detected interaction of SATB1 and CDP in several different tissue extracts. Association of purified SATB1 and CDP in vitro resulted in the inability of each protein to bind to DNA in gel retardation assays. CDP overexpression in cultured T cells led to a loss of detectable SATB1 binding to the MMTV promoter region, as measured by gel shift experiments. CDP overexpression also elevated MMTV long terminal repeat reporter gene activity in transient-transfection assays, a result consistent with neutralization of the SATB1 repressor function in T cells. SATB1 is very abundant in certain tissues, particularly thymus, whereas CDP is relatively ubiquitous, except in certain terminally differentiated cell types. Because of the tissue and cell type distribution of SATB1 and CDP, we propose that the SATB1-to-CDP ratio in different tissues is a novel mechanism for homeoproteins to control gene expression and differentiation in mammals.

CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

“…We have shown previously that MMTV expression is repressed by the homeodomain protein SATB1, and we have proposed that SATB1 restricts MMTV expression in many tissues other than the mammary gland, particularly T cells (40,41). Our studies also have shown that SATB1 binding activity for the MMTV NREs is absent in mammary gland tissue, as well as in cultured mammary cell lines (13,41).…”

“…Because all of the cultured cell lines that we have examined contain endogenous CDP that might interfere with the assay (our unpublished observations), the amount of pLC-LUC used in this experiment is critical for our ability to observe effects on CDP overexpression. To determine the optimal amount of pLC-LUC for these experiments, we transfected different amounts of pLC-LUC (20,40, and 60 g) into HC11 cells with 30 g of the CDP expression vector and 5 g FIG. 1.…”

“…To localize all of the CDP binding sites, we performed a series of DNase I footprinting assays with bacterially expressed purified CDP (40). Because full-length CDP is insoluble, an N-terminal deletion that removed the coiled-coil and CR1 domains of CDP (39) was used for footprinting with an endlabeled 120-bp fragment from the pNRE (Fig.…”

“…Polyclonal rabbit antibodies against glutathione S-transferase (GST)-SATB1 were kindly provided by Paul Gottlieb (University of Texas at Austin). Other polyclonal antibodies against GST-SATB1 or GST-CDP (CR2-Cterm) (39) were prepared by immunization of rabbits in accordance with the standard procedures of Cocalico Biologicals, Reamstown, Pa. Antibodies were shown to be specific for CDP or SATB1 as described previously (40). Western blots were prepared by lysis of cells in RIPA buffer (25 mM Tris-HCl [pH 7.8], 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 0.5% deoxycholate, 0.1% SDS) (23).…”

“…MMTV expression is suppressed in undeveloped mammary tissue but is activated in the lactating mammary gland (34). CDP also binds to the MMTV transcriptional control region (40,41). Therefore, it is possible that CDP functions as a repressor to suppress MMTV expression in undeveloped breast tissue, and viral suppression is relieved when mammary cells are fully differentiated.…”