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
The selective and temporal DNA methylation plays an important role in the self-renewal and differentiation of hematopoietic stem cells (HSCs), but the molecular mechanism that controls the dynamics of DNA methylation is not understood. Here, we report that the PIAS1 epigenetic pathway plays an important role in regulating HSC self-renewal and differentiation. PIAS1 is required for maintaining the quiescence of dormant HSCs and the long-term repopulating capacity of HSC. Pias1 disruption caused the abnormal expression of lineage-associated genes. Bisulfite sequencing analysis revealed the premature promoter demethylation of Gata1, a key myeloerythroid transcription factor and a PIAS1-target gene, in Pias1(-/-) HSCs. As a result, Pias1 disruption caused the inappropriate induction of Gata1 in HSCs and common lymphoid progenitors (CLPs). The expression of other myeloerythroid genes was also enhanced in CLPs and lineage-negative progenitors, with a concurrent repression of B cell-specific genes. Consistently, Pias1 disruption caused enhanced myeloerythroid, but reduced B lymphoid lineage differentiation. These results identify a novel role of PIAS1 in maintaining the quiescence of dormant HSCs and in the epigenetic repression of the myeloerythroid program.
Subject(s)
Cell Differentiation/genetics , Cell Proliferation , Hematopoietic Stem Cells/physiology , Protein Inhibitors of Activated STAT/physiology , Animals , Bone Marrow Cells/physiology , Cell Lineage/genetics , Cell Movement/genetics , Cellular Microenvironment/genetics , Epigenesis, Genetic , Mice , Mice, Inbred C57BL , Mice, Knockout , Stem Cell Niche/geneticsABSTRACT
CD4(+)Foxp3(+) regulatory T (T(reg)) cells are important for maintaining immune tolerance. Understanding the molecular mechanism that regulates T(reg) differentiation will facilitate the development of effective therapeutic strategies against autoimmune diseases. We report here that the SUMO E3 ligase PIAS1 restricts the differentiation of natural T(reg) cells by maintaining a repressive chromatin state of the Foxp3 promoter. PIAS1 acts by binding to the Foxp3 promoter to recruit DNA methyltransferases and heterochromatin protein 1 for epigenetic modifications. Pias1 deletion caused promoter demethylation, reduced histone H3 methylation at Lys(9), and enhanced promoter accessibility. Consistently, Pias1(-/-) mice displayed an increased natural T(reg) cell population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of natural T(reg) cells.
Subject(s)
Epigenesis, Genetic , Lymphopoiesis/genetics , Protein Inhibitors of Activated STAT/physiology , Repressor Proteins/physiology , T-Lymphocytes, Regulatory/cytology , Ubiquitin-Protein Ligases/physiology , Animals , Binding Sites , CD4-Positive T-Lymphocytes/cytology , Chromatin/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , DNA Methyltransferase 3A , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Forkhead Transcription Factors/genetics , Histones/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Promoter Regions, Genetic , T-Lymphocytes, Regulatory/immunology , DNA Methyltransferase 3BABSTRACT
RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.
Subject(s)
Antigens, Neoplasm/metabolism , Centromere/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Molecular Chaperones/metabolism , Nuclear Pore Complex Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Anaphase , Aneuploidy , Animals , Carcinogens , Fibroblasts/cytology , Fibroblasts/metabolism , Mice , Mice, Knockout , Mitosis , Molecular Chaperones/genetics , Mutation , Neoplasms/chemically induced , Neoplasms/metabolism , Nuclear Pore Complex Proteins/genetics , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
NF-kappaB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-kappaB/STAT signaling have been extensively studied, how NF-kappaB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-kappaB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-kappaB and STAT1-dependent genes in response to LPS or IFN-gamma treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-kappaB/STAT1 signaling. Pias1-/-Piasy-/- embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy-/- background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-kappaB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-kappaB/STAT1-mediated gene activation.
Subject(s)
Down-Regulation/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Inhibitors of Activated STAT/physiology , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Animals , Cells, Cultured , Gene Expression Regulation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/antagonists & inhibitors , NF-kappa B/physiology , Protein Inhibitors of Activated STAT/deficiency , Protein Inhibitors of Activated STAT/genetics , Repressor Proteins/genetics , Repressor Proteins/physiology , STAT1 Transcription Factor/antagonists & inhibitors , Transcriptional ActivationABSTRACT
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.
