The NF-B family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-B activity is critical, since abnormal NF-B signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 ( and bacterial lipopolysaccharide (LPS), activate the NF-B signaling pathway. NF-B is a family of dimeric transcription factors composed of members of the Rel family of DNA binding proteins, including NF-B1 (p50 and its precursor p105), NF-B2 (p52 and its precursor p100), c-Rel, RelA (p65), and RelB (11, 18). Upon stimulation, NF-B translocates into the nucleus, where it binds to specific DNA sequences and regulates transcription. NF-B is involved in mediating a wide spectrum of cellular responses, including infections, inflammation, and apoptosis (2, 27). Inappropriate regulation of NF-B is involved in a wide range of human diseases, including cancer, neurodegenerative disorders, arthritis, asthma, and chronic inflammation (3,4,10,12). The NF-B signaling pathway is tightly modulated at various levels by distinct regulatory proteins. For example, the binding of the IB family of proteins prevents the nuclear translocation of NF-B (16). However, a protein factor that can regulate the DNA binding activity of NF-B has not been documented.The PIAS (protein inhibitor of activated STAT) family of proteins consists of four members: PIAS1, PIAS3, PIASx, and PIASy (33). Members of the PIAS family have been suggested to regulate STAT-mediated transcription. Upon cytokine stimulation, PIAS binds to STAT and inhibits STAT-mediated gene activation (1,8,21,22). Among the PIAS family, PIAS1 and PIASy have been shown to inhibit STAT1-dependent transcription through distinct mechanisms. PIAS1 inhibits the transcriptional activity of STAT1 by blocking the DNA binding activity of STAT1. In contrast, PIASy does not affect the DNA binding activity of STAT1. It has been suggested that PIASy may act as a transcriptional corepressor of STAT1. The PIAS family of proteins has also been suggested to regulate a number of other transcription factors, including nuclear hormone receptors (13,28,36,37), LEF1 (31), and p53 (15,26,32).To understand the physiological role of PIAS1, we have recently generated Pias1 null mice (23). Detailed gene activation analysis indicates that PIAS1 selectively regulates a subset of interferon (IFN)-inducible genes. The antiviral activity of IFNs is significantly enhanced by Pias1 disruption. In addition, Pias1 null mice show enhanced protection against pathogenic infection. These results support a physiological role of PIAS1 in the negative regulation of IFN-activated STAT1-mediated gene activation and demonstrate an important role of PIAS1 in innate immune responses.Since STAT1 and the Rel family of proteins share structural similarity in their DNA binding domains (6), we explored the possible involvement of PIAS1 in the regulation of NF-B. Here we report that PIAS1 interacts with ...

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.

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.

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.