THE MATRIX ATTACHMENT REGION-BINDING PROTEIN SATB1 PARTICIPATES IN NEGATIVE REGULATION OF TISSUE-SPECIFIC GENE-EXPRESSION

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing, Tn parti cular, transcriptional regulation is believed to be accomplished by bi nding of chromatin loops to the nuclear matrix and by the concentratio n of specific transcription factors near these matrix attachment regio ns (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription, Recent ly, we have identified two cellular protein complexes (NBP and UBP) th at bind to a region of the mouse mammary tumor virus (MMTV) long termi nal repeat (LTR) previously shown to contain at least two negative reg ulatory elements (NREs) termed the promoter-proximal and promoter-dist al NREs, These NREs are absent from MMTV strains that cause T-cell lym phomas instead of mammary carcinomas, We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promote r-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter a nd destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thym ic nuclear extracts, we obtained a single major protein of 115 kDa tha t was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-bindin g protein 1 (SATB1), Antibody ablation, distamycin inhibition of bindi ng, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1, Similar types of experiments were used to show that the UBP complex contained the h omeodomain protein Cux/CDP that binds the MAR of the intronic heavy-ch ain immunoglobulin enhancer, By using the p924 mutation within the MMT V LTR upstream of the chloramphenicol acetyltransferase gene, we gener ated two strains of transgenic mice that had a dramatic elevation of r eporter gene expression in lymphoid tissues compared with reporter gen e expression in mice expressing wild-type LTR constructs, Thus, the 92 4 mutation in the SATB1-binding site dramatically elevated MMTV transc ription in lymphoid tissues, These results and the ability of the prox imal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonst rate the role of MAR-binding proteins in tissue-specific gene regulati on and in MMTV-induced oncogenesis.