Nuclear matrix binding regulates SATB1-mediated transcriptional repression.
Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.
Seo
Jin
J
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.
Lozano
Mary M
MM
Dudley
Jaquelin P
JP
D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit; D007438:Introns; D019169:Jurkat Cells; D008024:Ligands; D008321:Mammary Glands, Animal; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D036961:Matrix Attachment Region Binding Proteins / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D018123:Receptors, Interleukin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012190:Retroviridae / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D013347:Subcellular Fractions; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; C508017:IL2RA protein, human; C508018:Il2ra protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D008024:Ligands; D036961:Matrix Attachment Region Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D011965:Receptors, Glucocorticoid; D018123:Receptors, Interleukin; D011993:Recombinant Fusion Proteins; C076189:SATB1 protein, human; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D012260:Ribonucleases