IFN-␥ treatment of cells leads to tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 1 followed by dimerization through a reciprocal Src homology 2-phosphotyrosine interaction near the -COOH end of each monomer, forming a parallel structure that accumulates in the nucleus to drive transcription. Prompt dephosphorylation and return to the cytoplasm completes the activation-inactivation cycle. Nonphosphorylated STATs dimerize, and a previously described interface between N-terminal domain (ND) dimers has been implicated in this dimerization. A new crystal structure of nonphosphorylated STAT1 containing the ND dimer has two possible configurations for the body of STAT1, one of which is antiparallel. In this antiparallel structure, the Src homology 2 domains are at opposite ends of the dimer, with the coiled:coil domain of one monomer interacting reciprocally with the DNA-binding domain of its partner. Here, we find that mutations in either the coiled:coil͞DNA-binding domain interface or the ND dimer interface block dimerization of nonphosphorylated molecules and cause a resistance to dephosphorylation in vivo and resistance to a tyrosine phosphatase in vitro. We conclude that a parallel STAT1 phosphodimer not bound to DNA most likely undergoes a conformational rearrangement (parallel to antiparallel) to present the phosphotyrosine efficiently for dephosphorylation.dephosphorylation ͉ structural rearrangement S ignal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that can be activated by a variety of tyrosine kinases in response to many different cytokine, growth factor, and peptide ligands binding to their respective cell surface receptors. Accumulation in the nucleus of tyrosine phosphorylated STAT dimers is followed by DNA binding, activation of target gene transcription, dephosphorylation, and return to the cytoplasm (1). The crystal structure of the phosphodimer core (amino acids 130-712), bound to DNA, showed a reciprocal phosphotyrosine (pY)-Src homology 2 (SH2) interaction at one end of a parallel dimer (2), with the DNA separating the monomers along the long axis of the monomers. Mao et al. (3) have now described a dimeric structure of nonphosphorylated STAT1, including the core (amino acids 130-712), as well as the core plus the N-terminal domain (ND) (amino acids 1-683). (The terminal 29 aa, 684-712, in the core were unstructured and omitted from study.) The body of each of the monomers in the nonphosphorylated structure is identical to the monomers in the previously reported phosphorylated dimer (2). However, the monomers in the nonphosphorylated core structure are arranged differently in space. In contrast to the dimeric, parallel structure of the phosphorylated STAT1, the SH2 domains in the nonphosphorylated core structure project from the opposite ends of an antiparallel dimer with a dimeric interface formed by reciprocal contacts between the coiled:coil (CC) and DNA-binding domains (DBD) of the monomers. In the crystal...

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The STAT1 transcription factor is organized into several highly conserved domains, each of which has been assigned a function with the exception of the linker domain. We previously characterized a mutant in the linker domain of STAT1 that gave normal DNA binding using a standard probe in an electrophoretic mobility assay but failed to activate transcription in response to interferon ␥. We now report the mechanistic basis for the inactivity of this STAT1(K544A/E545A) mutant. Rather than failing to attract transcriptional coactivators, the STAT1(K544A/E545A) mutant has a subtle biophysical defect, which prevents accumulation of the activated protein on chromatin in vivo: the mutant has comparable K d with greatly increased k off for DNA binding. The increase in both on-rate and off-rate of DNA binding results in a substantially reduced residence time of STAT1(K544A/E545A) on STAT binding sites. We find a similar correlation between off-rate and transcriptional potency for STAT1(N460A), which bears a mutation in the DNA binding domain. These results yield insight into the rate of complex assembly involving STAT1 that leads to transcriptional stimulation.

Background: Signal transducer and activator of transcription (STAT) activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results: Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1). Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFN)γ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II) occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS)-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD) of Pol II are disrupted during gene activation as well.

Previously unrecognized mRNAs originating from a dual promoter at the stat92E locus are described. One of these encodes a truncated protein, ⌬NSTAT92E, that lacks the N-terminal 133 amino acids. Antibodies detect both the full-length and truncated molecules early in embryogenesis (1-5 h), and mRNA detection by specific RT-PCR reactions accords with the protein distribution. Given that the N termini of mammalian STATs are known to have positive functions in transcriptional activation, we explored the role of ⌬NSTAT92E early in embryogenesis. By increasing the ⌬NSTAT92E-to-STAT92E ratio in overexpression and RNAi experiments, we observe phenotypes compatible with suppression of wild-type STAT92E activity. We therefore conclude that the short form of STAT92E is a naturally occurring dominant-negative product that can be added to the growing list of negative regulators of STAT activity.

The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway drives cellular growth, differentiation, and the immune response. STAT-activated gene expression is both rapid and transient and requires dynamic post-translational modification of the chromatin template. We previously showed that monoubiquitination of histone H2B (ubH2B) is highly dynamic at the STAT1 target gene, interferon regulatory factor 1 (IRF1), suggesting that a deubiquitinase is recruited during gene activation. Here, we report that RNAi-mediated knockdown of the ubiquitin hydrolase, USP22, results in 2-fold higher ubH2B, and 2-fold lower transcriptional elongation at IRF1. We also demonstrate that USP22 depletion diminishes 3'-end cleavage/polyadenylation by 2- to 3-fold. Furthermore, the polyadenylation factor CPSF73 is not effectively recruited, and serine 2 phosphorylation (Ser2P) of the C-terminal domain of RNA polymerase II is also disrupted. The transcriptional and processing defects observed in the USP22-knockdown cells are reversed by transient USP22 overexpression. Together, these results suggest that ubH2B helps recruit polyadenylation factors to STAT1-activated genes. We propose a working model, wherein a cycle of H2B ubiquitination/deubiquitination specifies Ser2P to regulate elongation and 3'-end processing of JAK-STAT-inducible mRNAs. These results further elaborate USP22 function and its role as a putative cancer stem cell marker.

The ubiquitin hydrolase, USP22 contributes to 3'-end processing of JAK-STAT inducible genes.

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