Novel role of the RET finger protein in estrogen receptor-mediated transcription in MCF-7 cells.

The Scaffold attachment factor B1 (SAFB1) is an estrogen receptor (ESR1) repressor that has been proposed to inhibit breast tumorigenesis. To obtain insight into the functions of SAFB1 we utilized a yeast two-hybrid screen and identified the Ret finger protein (RFP) as interacting with the SAFB1 C-terminus. RFP is a member of the trimotif (TRIM) family of proteins, which we found widely expressed in a series of breast cancer cell lines. We confirmed the interaction between SAFB1 and RFP through in vitro (GST-pull-down) and in vivo (coimmunoprecipitations) assays. We hypothesized that SAFB1 functions as a scaffolding protein to recruit proteins such as RFP into proximity with ESR1. Consequently, we asked whether RFP would modulate ESR1 activity and we discovered that RFP was important for the ESR1-dependent expression of cyclin D1 (CCND1) and the progesterone receptor (PR), but not IRS1 or MYC. Although RFP did not interact with ESR1 directly, it does coimmunoprecipitate with ESR1, demonstrating that RFP is found within the same protein complex. Chromatin immunoprecipitation assays (ChIP) located RFP to the TFF1 promoter, a known ESR1-regulated gene. Taken together, our study provides further evidence that coactivation and corepression are integrally linked processes and that RFP is a component of an ESR1 regulatory complex.

Disruption of scaffold attachment factor B1 leads to TBX2 up-regulation, lack of p19ARF induction, lack of senescence, and cell immortalization.

Scaffold attachment factor B1 (SAFB1) is a multifunctional protein, which has previously been implicated in breast cancer. Here, we show that genetic deletion of SAFB1 in mouse embryonic fibroblasts (MEF) leads to spontaneous immortalization and altered expression of two proteins involved in immortalization and escape from senescence: low levels of p19(ARF) and high levels of TBX2. Inactivation of TBX2 using a dominant-negative TBX2 resulted in up-regulation of p19(ARF) in SAFB1 knockout MEFs. SAFB1 loss also caused lack of contact inhibition, increased foci formation, and increased oncogene-induced anchorage-independent growth. These findings suggest that SAFB1 is a novel player in cellular immortalization and transformation.

Scaffold attachment factor SAFB1 suppresses estrogen receptor alpha-mediated transcription in part via interaction with nuclear receptor corepressor.

Activity of the estrogen receptor (ER) is regulated through interaction with coactivators and corepressors. These proteins are present in large complexes, suggesting functional interactions among various cofactors. Scaffold attachment factors B1 and B2 (SAFB1/2) and nuclear receptor corepressor (N-CoR) function as ERalpha corepressors--they directly interact with ERalpha, and repress transcription via repression domains. We asked the question whether SAFB1/2 and N-CoR could directly interact with each other, and whether this interaction results in altered repressive activities. Employing coimmunoprecipitation, cofractionation, and colocalization experiments, we have shown that SAFB1/2 interact with the nuclear receptor corepressor N-CoR. This interaction was direct, and was mediated in vitro and in vivo through the C-terminal region of SAFB1 (amino acids 600-915 and the N-terminal region of N-CoR (amino acids 1-373)). Decrease of SAFB1 or N-CoR expression by small interfering RNA resulted in an increase of the estrogen response in reporter assays, confirming prior data that both proteins are attenuating estrogen-mediated induction of genes. Importantly, the effect of SAFB1 on this attenuation was significantly decreased in the presence of N-CoR small interfering RNA. Using chromatin immunoprecipitation assays, we observed that SAFB1/2 and N-CoR were recruited to the pS2 promoter in the absence of estrogen, and this recruitment was enhanced in the presence of Tamoxifen. Detailed kinetic studies showed that the addition of estrogen resulted in the concurrent release of SAFB1/2 and N-CoR from the promoter. Finally, we measured expression of SAFB1/2 and N-CoR in 289 clinical breast cancer specimens, and detected a strong and highly significant correlation between their expression levels. Taken together, our studies demonstrate that SAFB1/2 and N-CoR interact, and that this interaction is, at least in part, necessary for SAFB1's repressive activities. The coexpression of these proteins in breast cancer specimens, and the combined recruitment (and release) of SAFB1/2 and N-CoR furthermore suggests that this interaction has functional relevance.

Scaffold attachment factor B1 functions in development, growth, and reproduction.

Scaffold attachment factor B1 (SAFB1) is a multifunctional protein that can bind both DNA and RNA and is involved in RNA processing and stress response. In addition, SAFB1 contains a transcriptional repression domain and can bind certain hormone receptors and repress their activity. To assess the role of SAFB1 in vivo, we generated SAFB1 mutant mice through targeted deletion in embryonic stem cells. While viable homozygous mutant (SAFB1-/-) mice were obtained, genotypic distribution indicated that homozygous deficiency resulted in both prenatal and neonatal lethality. Mice lacking SAFB1 exhibited dwarfism, as a result of in utero growth retardation, and had low serum insulin-like growth factor 1 (IGF1) levels. In agreement with the previous characterization of SAFB1 as a corepressor for hormone receptors, we found that SAFB1-/- mice displayed dramatic defects in the development and function of the reproductive system. Male SAFB1 null mice were infertile, apparently because of low circulating levels of testosterone. SAFB1-/- testes were small and showed progressive degeneration of the germinal epithelium, increased apoptosis of germ cells, and Leydig cell hyperplasia. SAFB-/- female mice were subfertile and showed progressive infertility, in part because of defects in oviductal transport and reduced numbers of follicles. Immortalized SAFB1-/- mouse embryonic fibroblasts showed cell-intrinsic defects including increased transcriptional estrogen receptor alpha activity and enhanced responsiveness to IGF1. Together, these in vivo findings establish a critical role for SAFB1 in development, growth regulation, and reproduction.

Structure-function analysis of the estrogen receptor alpha corepressor scaffold attachment factor-B1: identification of a potent transcriptional repression domain.

Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ERalpha)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERalpha activity. SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ERalpha activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.

Estrogen-mediated down-regulation of E-cadherin in breast cancer cells.

E-cadherin is an important mediator of cell-cell interactions, and has been shown to play a crucial role in breast tumor suppression. Its inactivation occurs through instability at its chromosomal locus and mutations, but also through epigenetic mechanisms such as promoter hypermethylation and transcriptional silencing. We show here that the potent mitogen estrogen causes down-regulation of E-cadherin levels in both normal and tumorigenic breast epithelial cells, and that this down-regulation is reversed by antiestrogens. The reduction in E-cadherin levels is via a decrease in promoter activity and subsequent mRNA levels. Chromatin immunoprecipitation assays revealed that estrogen receptor and corepressors were bound to the E-cadherin promoter, and that overexpression of corepressors such as scaffold attachment factor B resulted in enhanced repression of E-cadherin. We propose that estrogen-mediated down-regulation of E-cadherin is a novel way of reducing E-cadherin levels in estrogen receptor-positive breast cancer.

SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor.

We have characterized previously the nuclear matrix protein/scaffold attachment factor (SAFB) as an estrogen receptor corepressor and as a potential tumor suppressor gene in breast cancer. A search of the human genome for other potential SAFB family members revealed that KIAA00138 (now designated as SAFB2) has high homology to SAFB (now designated as SAFB1). SAFB1 and SAFB2 are mapped adjacent to each other on chromosome 19p13.3 and are arranged in a bidirectional divergent configuration (head to head), being separated by a short (<500 bp) GC-rich intergenic region that can function as a bidirectional promoter. SAFB1 and SAFB2 share common functions but also have unique properties. As shown previously for SAFB1, SAFB2 functions as an estrogen receptor corepressor, and its overexpression results in inhibition of proliferation. SAFB1 and SAFB2 interact directly through a C-terminal domain, resulting in additive repression activity. They are coexpressed in a number of tissues, but unlike SAFB1, which is exclusively nuclear, SAFB2 is found in the cytoplasm as well as the nucleus. Consistent with its cytoplasmic localization, we detected an interaction between SAFB2 and vinexin, a protein involved in linking signaling to the cytoskeleton. Our findings suggest that evolutionary duplication of the SAFB gene has allowed it to retain crucial functions, but also to gain novel functions in the cytoplasm and/or nucleus.