The nuclear matrix affects SATB1-mediated MMTV suppression

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary adenocarcinomas in susceptible mouse strains. Milk-borne MMTV initially causes a low level infection of gut-associated lymphoid cells, which then deliver the virus to mammary epithelial cells, a target for high levels of MMTV replication and insertional mutagenesis. Although wild-type MMTV is not lymphomagenic, variants that cause thymic lymphomas have alterations in the long terminal repeat (LTR), including a deletion of negative regulatory elements (NREs). This deleted region contains binding sites for at least two cellular proteins, CCAAT displacement protein (CDP) and special AT-rich binding protein 1 (SATB1). SATB1 is expressed primarily in the thymus and has been proposed to suppress MMTV expression and integration in lymphoid tissues. Because of previous difficulties with SATB1 overexpression in culture, a transcriptional assay system for SATB1 function was developed. Rat fibroblasts or mouse mammary cells, which express little SATB1, were stably transfected with MMTV-luciferase reporter genes. Transduction with retroviruses expressing wild-type SATB1 suppressed MMTV LTR expression. SATB1 transduction also suppressed expression of integrated MMTV proviruses in rat cells, consistent with the primarily repressor function of this transcription factor. To delineate domains responsible for DNA binding and suppression of MMTV transcription in this system, SATB1 mutants were generated. SATB1 is known to bind to the nuclear matrix, which has been shown to play important roles in transcription. A nuclear matrix targeting sequence (NMTS) of SATB1 was identified as a 55-amino acid region between the dimerization domain and the major DNA-binding domain. Both the DNA-binding and dimerization domains were necessary for binding to the MMTV NRE using gel shift assays, but the NMTS was not. Deletion of the DNA-binding domain or the NMTS significantly alleviated repression of stably integrated MMTV LTR-reporter genes, confirming the importance of both DNA binding and nuclear matrix binding for SATB1-mediated MMTV suppression. Additionally, cellular factors interacting with this SATB1 NMTS were identified through screening of a human thymic cDNA library using the yeast two-hybrid system. Further studies using this reporter system for SATB1 function and identification of protein interactions with the nuclear matrix should provide unique insights into transcriptional regulation and tissue-specific transcription.