Abstract
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.
Highlights
Mouse mammary tumor virus (MMTV)1 transcription is controlled by regions largely found within the U3 region of the long
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
To determine whether nuclear matrix targeting of SATB1 is important for transcriptional repression of the MMTV LTR, we constructed a series of truncation/deletion mutants in an overexpression vector (Fig. 1A)
Summary
Mouse mammary tumor virus (MMTV) transcription is controlled by regions largely found within the U3 region of the long. MMTV transcription is suppressed in specific tissues by the presence of several negative regulatory elements (NREs) located upstream of the HRE in the viral LTR (6 –10). CDP overexpression in mammary or fibroblastic cells results in suppression of MMTV LTR-reporter gene expression both in the presence and absence of glucocorticoids [28, 29, 31]. MMTV RNA expression is highest in the lactating mammary gland, a tissue in which CDP DNA-binding activity for the MMTV LTR is undetectable [28]. Loss of CDP-binding sites within the NREs elevates MMTV transcription in the mammary gland and accelerates tumorigenesis [32]. The transcription factor, SATB1, binds to the MMTV NRE within MAR sequences (9 –11) and recognizes a specific sequence context that exhibits a high base-unpairing or -unwinding propensity [9, 25]. SATB1 is most abundant in the thymus [25], a tissue that is semi-permissive for
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