ABSTRACT
Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast/pathology , Carcinogenesis/genetics , Epigenesis, Genetic/genetics , Protein Inhibitors of Activated STAT/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Animals , Cell Line, Tumor , Cyclin D2/genetics , DNA Methylation/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Gene Silencing , Humans , Mice, SCID , Proto-Oncogene Proteins/genetics , Receptors, Estrogen/genetics , Ubiquitin-Protein Ligases/genetics , Wnt Proteins/genetics , Wnt-5a ProteinABSTRACT
How inflammatory stimuli signal to the nucleus to restrict inflammation is poorly understood. Protein inhibitor of activated STAT1 (PIAS1), a transcriptional regulator that possesses small ubiquitin-related modifier (SUMO) E3 ligase activity, inhibits immune responses by selectively blocking the binding of NF-kappaB and STAT1 to gene promoters. We report here that PIAS1 becomes rapidly phosphorylated on Ser90 residue in response to various inflammatory stimuli. Mutational studies indicate that Ser90 phosphorylation is required for PIAS1 to repress transcription. Upon TNF treatment, wild-type PIAS1, but not the Ser90A mutant, becomes rapidly associated with the promoters of NF-kappaB target genes. Furthermore, IKKalpha, but not IKKbeta, interacts with PIAS1 in vivo and mediates PIAS1 Ser90 phosphorylation, a process that requires the SUMO ligase activity of PIAS1. Our results identify a signaling pathway in which proinflammatory stimuli activate the IKKalpha-mediated sumoylation-dependent phosphorylation of PIAS1 for the immediate repression of inflammatory gene activation.
Subject(s)
I-kappa B Kinase/metabolism , Inflammation/immunology , Protein Inhibitors of Activated STAT/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Gene Expression , Humans , I-kappa B Kinase/genetics , Ligands , Macrophages/immunology , Macrophages/metabolism , Mice , Phosphorylation , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Transcription, GeneticABSTRACT
The NF-kappaB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-kappaB activity is critical, since abnormal NF-kappaB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-kappaB. Upon cytokine stimulation, the p65 subunit of NF-kappaB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-kappaB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-kappaB-regulated genes is significantly enhanced in Pias1-/- cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-kappaB-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-kappaB.
Subject(s)
Cell Nucleus/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Transcription, Genetic/genetics , Active Transport, Cell Nucleus/physiology , Animals , Bone Marrow/metabolism , Cells, Cultured , Chromatin Immunoprecipitation , Cytokines/metabolism , Electrophoretic Mobility Shift Assay , Enzyme Activation/physiology , Humans , Macrophages/metabolism , Mice , Mice, Knockout , Microarray Analysis , Protein Inhibitors of Activated STAT , Protein Serine-Threonine Kinases/genetics , Signal Transduction/physiology , Transcription Factor RelA , NF-kappaB-Inducing KinaseABSTRACT
PIASy, a member of the protein inhibitor of activated STAT (PIAS) family, represses the transcriptional activity of the androgen receptor (AR). In this report, we investigate the mechanism of PIASy-mediated repression of AR. We show that AR binds to the RING-finger like domain of PIASy. PIASy contains two transcriptional repression domains, RD1 and RD2. RD1, but not RD2, is required for PIASy-mediated repression of AR. We show that the RD1 domain binds HDAC1 and HDAC2 and that HDAC activity is required for PIASy-mediated AR repression. PIAS proteins possess small ubiquitin-related modifier (SUMO) E3 ligase activity. Conjugation of SUMO-1 to AR has been implicated in the regulation of AR activity. We examine if the SUMO ligase activity of PIASy is required for PIASy to repress AR. We show that a mutant PIASy, defective in promoting sumoylation, retains the ability to repress AR transcription. In addition, mutation of all the known sumoylation acceptor sites of AR does not affect the transrepression activity of PIASy on AR. Our results suggest that PIASy may repress AR by recruiting histone deacetylases, independent of its SUMO ligase activity.
