ABSTRACT
Scaffold attachment factors SAFB1 and SAFB2 are multifunctional proteins that share >70% sequence similarity. SAFB1-knockout (SAFB1(-/-)) mice display a high degree of lethality, severe growth retardation, and infertility in male mice. To assess the in vivo role of SAFB2, and to identify unique functions of the two paralogs, we generated SAFB2(-/-) mice. In stark contrast to SAFB1(-/-), SAFB2(-/-) offspring were born at expected Mendelian ratios and did not show any obvious defects in growth or fertility. Generation of paralog-specific antibodies allowed extensive expression analysis of SAFB1 and SAFB2 in mouse tissues, showing high expression of both SAFB1 and SAFB2 in the immune system, and in hormonally controlled tissues, with especially high expression of SAFB2 in the male reproductive tract. Further analysis showed a significantly increased testis weight in SAFB2(-/-) mice, which was associated with an increased number of Sertoli cells. Our data suggest that this is at least in part caused by alterations in androgen-receptor function and expression upon deletion of SAFB2. Thus, despite a high degree of sequence similarity, SAFB1(-/-) and SAFB2(-/-) mice do not totally phenocopy each other. SAFB2(-/-) mice are viable, and do not show any major defects, and our data suggest a role for SAFB2 in the differentiation and activity of Sertoli cells that deserves further study.
Subject(s)
Carrier Proteins/genetics , DNA-Binding Proteins/genetics , RNA-Binding Proteins/genetics , Animals , Body Weight , Carrier Proteins/physiology , Cell Differentiation , DNA-Binding Proteins/physiology , Disease Models, Animal , Exons , Female , Gene Deletion , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , RNA-Binding Proteins/physiology , Reproduction , Sertoli Cells/cytology , Signal TransductionABSTRACT
Transcription of the HER2 oncogene can be repressed by estrogen (E2). We now show that, a splice isoform of the nuclear receptor coactivator AIB1, AIB1-Δ4, is able to reverse E2 repression of HER2 gene expression in breast cancer cells. The first 224 amino acids of AIB1 that are absent in AIB1-Δ4, bind a co-repressor, ANCO1. Using chromatin immunoprecipitation assay approaches in MCF7 and BT474 cell lines, we demonstrate that AIB1 and AIB1-Δ4 can bind to the E2 regulatory site in the first intron of the HER2 gene, after E2 treatment, but only full-length AIB1 recruits ANCO1. Consistent with E2-induced chromatin repression, the AIB1-ANCO1 complex recruits HDAC3 and HDAC4 to the intronic estrogen response element and the proximal promoter acquires the repressive chromatin mark H3K9me3 and loses H3K4me1. In contrast, AIB1-Δ4 does not recruit ANCO 1, HDAC3, or HDAC4 and the proximal promoter retains activation marks of H3K4me1. In cell lines with low levels of ANCO1 (T47D), E2 does not repress HER2 gene transcription but the repressive response can be restored by overexpression of ANCO1. ANCO1 can also repress other E2-responsive genes, indicating that AIB1, AIB1-Δ4 and ANCO1 are important determinants of endocrine and growth factor responsiveness in breast cancer.
Subject(s)
Gene Expression Regulation, Neoplastic , Nuclear Receptor Coactivator 3/physiology , Receptor, ErbB-2/genetics , Repressor Proteins/physiology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chromatin Assembly and Disassembly , Estradiol/physiology , Female , HEK293 Cells , Histone Deacetylases/metabolism , Humans , MCF-7 Cells , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/metabolism , Oncogenes , Protein Binding , Receptor, ErbB-2/metabolism , Response Elements , Signal Transduction , Transcription, GeneticABSTRACT
Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)-positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.
Subject(s)
Breast Neoplasms/genetics , Cellular Reprogramming/genetics , Drug Resistance, Neoplasm/genetics , Epigenesis, Genetic/genetics , Estrogens/pharmacology , Homeodomain Proteins/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Aromatase Inhibitors/pharmacology , Aromatase Inhibitors/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cellular Reprogramming/drug effects , DNA Methylation/drug effects , DNA Methylation/genetics , Drug Resistance, Neoplasm/drug effects , Epigenesis, Genetic/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing/drug effects , Histones/metabolism , Homeodomain Proteins/metabolism , Humans , MCF-7 Cells , Mice , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Promoter Regions, Genetic , Xenograft Model Antitumor AssaysABSTRACT
The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator that is overexpressed in various types of human cancers. However, the molecular mechanisms controlling AIB1 expression in the majority of cancers remain unclear. In this study, we identified a novel interacting protein of AIB1, forkhead-box protein G1 (FoxG1), which is an evolutionarily conserved forkhead-box transcriptional corepressor. We show that FoxG1 expression is low in breast cancer cell lines and that low levels of FoxG1 are correlated with a worse prognosis in breast cancer. We also demonstrate that transient overexpression of FoxG1 can suppress endogenous levels of AIB1 mRNA and protein in MCF-7 breast cancer cells. Exogenously expressed FoxG1 in MCF-7 cells also leads to apoptosis that can be rescued in part by AIB1 overexpression. Using chromatin immunoprecipitation, we determined that FoxG1 is recruited to a region of the AIB1 gene promoter previously characterized to be responsible for AIB1-induced, positive autoregulation of transcription through the recruitment of an activating, multiprotein complex, involving AIB1, E2F transcription factor 1, and specificity protein 1. Increased FoxG1 expression significantly reduces the recruitment of AIB1, E2F transcription factor 1 and E1A-binding protein p300 to this region of the endogenous AIB1 gene promoter. Our data imply that FoxG1 can function as a pro-apoptotic factor in part through suppression of AIB1 coactivator transcription complex formation, thereby reducing the expression of the AIB1 oncogene.
Subject(s)
Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Forkhead Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Receptor Coactivator 3/genetics , Repressor Proteins/metabolism , Transcription, Genetic , Apoptosis/genetics , Down-Regulation/genetics , E2F1 Transcription Factor/metabolism , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , MCF-7 Cells , Models, Biological , Nuclear Receptor Coactivator 3/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Protein Stability , Sp1 Transcription Factor/metabolismABSTRACT
Sumoylation is an emerging modification associated with a variety of cellular processes including the regulation of transcriptional activities of nuclear receptors and their coregulators. As SUMO modifications are often associated with transcriptional repression, we examined if sumoylation was involved in modulation of the transcriptional repressive activity of scaffold attachment factor B1. Here we show that SAFB1 is modified by both the SUMO1 and SUMO2/3 family of proteins, on lysine's K231 and K294. Further, we demonstrate that SAFB1 can interact with PIAS1, a SUMO E3 ligase which mediates SAFB1 sumoylation. Additionally, SENP1 was identified as the enzyme desumoylating SAFB1. Mutation of the SAFB1 sumoylation sites lead to a loss of transcriptional repression, at least in part due to decreased interaction with HDAC3, a known transcriptional repressor and SAFB1 binding partner. In summary, the transcriptional repressor SAFB1 is modified by both SUMO1 and SUMO2/3, and this modification is necessary for its full repressive activity.
Subject(s)
Co-Repressor Proteins/metabolism , Matrix Attachment Region Binding Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Receptors, Estrogen/metabolism , Sumoylation , Amino Acid Sequence , Cell Line, Tumor , Co-Repressor Proteins/genetics , Consensus Sequence , Gene Expression Regulation , HEK293 Cells , Humans , Matrix Attachment Region Binding Proteins/genetics , Molecular Sequence Data , Nuclear Matrix-Associated Proteins/genetics , Protein Inhibitors of Activated STAT/metabolism , Receptors, Estrogen/genetics , SUMO-1 Protein , Small Ubiquitin-Related Modifier Proteins/metabolism , Transcription, Genetic , UbiquitinsABSTRACT
The examination of scaffold attachment factor B1 (SAFB1) and its multiple functions and tasks in cellular processes provides insight into its role in diseases, such as cancer. SAFB1 is a large multi-domain protein with well-described functions in transcriptional repression, and RNA splicing. It is ubiquitously expressed, and has been shown to be important in numerous cellular processes including cell growth, stress response, and apoptosis. SAFB1 is part of a protein family with at least two other family members, SAFB2 and the SAFB-like transcriptional modulator SLTM. The goal of this prospect article is to summarize known functions of SAFB1, and its roles in cellular processes, but also to speculate on less well described, novel attributes of SAFB1, such as a potential role in chromatin organization. This timely review shows aspects of SAFB1, which are proving to have a complexity far greater than was previously thought.
Subject(s)
DNA-Binding Proteins/physiology , Matrix Attachment Region Binding Proteins/physiology , Nuclear Matrix-Associated Proteins/physiology , RNA-Binding Proteins/physiology , Receptors, Estrogen/physiology , Animals , Apoptosis , Cell Proliferation , Chromatin/metabolism , Chromatin Assembly and Disassembly , Gene Expression Regulation , Humans , Matrix Attachment Region Binding Proteins/chemistry , Mice , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Matrix-Associated Proteins/chemistry , Organ Specificity , Protein Structure, Tertiary , Receptors, Estrogen/chemistry , Signal Transduction/physiology , Stress, Physiological/physiology , Transcription, GeneticABSTRACT
Activation of estrogen receptor alpha (ERalpha) results in both induction and repression of gene transcription; while mechanistic details of estrogen induction are well described, details of repression remain largely unknown. We characterized several ERalpha-repressed targets and examined in detail the mechanism for estrogen repression of Reprimo (RPRM), a cell cycle inhibitor. Estrogen repression of RPRM is rapid and robust and requires a tripartite interaction between ERalpha, histone deacetylase 7 (HDAC7), and FoxA1. HDAC7 is the critical HDAC needed for repression of RPRM; it can bind to ERalpha and represses ERalpha's transcriptional activity--this repression does not require HDAC7's deacetylase activity. We further show that the chromatin pioneer factor FoxA1, well known for its role in estrogen induction of genes, is recruited to the RPRM promoter, is necessary for repression of RPRM, and interacts with HDAC7. Like other FoxA1 recruitment sites, the RPRM promoter is characterized by H3K4me1/me2. Estrogen treatment causes decreases in H3K4me1/me2 and release of RNA polymerase II (Pol II) from the RPRM proximal promoter. Overall, these data implicate a novel role for HDAC7 and FoxA1 in estrogen repression of RPRM, a mechanism which could potentially be generalized to many more estrogen-repressed genes and hence be important in both normal physiology and pathological processes.