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. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, 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 homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.
Liu
J
J
Department of Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, 78712, USA.
Bramblett
D
D
Zhu
Q
Q
Lozano
M
M
Kobayashi
R
R
Ross
S R
SR
Dudley
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004214:Distamycins / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D036961:Matrix Attachment Region Binding Proteins; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors
D000998:Antiviral Agents; C080632:CUX1 protein, human; C000624101:Cux1 protein, mouse; D004268:DNA-Binding Proteins; D004214:Distamycins; D018398:Homeodomain Proteins; D036961:Matrix Attachment Region Binding Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C076189:SATB1 protein, human; D014157:Transcription Factors; C030000:stallimycin