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James E Darnell

Publications and source records attributed to James E Darnell.

At least 19 recordsLinked to original sources
PubMed2006

Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain.

We report experiments that infer a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, we show that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY-SH2 binding between the monomers of the dimer while ND ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation.

Binding Sites↗
PubMed2006

Cyclin D1 is transcriptionally regulated by and required for transformation by activated signal transducer and activator of transcription 3.

Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer-derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1(-/-) and cyclin D1(-/+) fibroblasts led to anchorage-independent growth of only cyclin D1(-/+) cells. Furthermore, knockdown of cyclin D1 in breast carcinoma cells led to a reduction in anchorage-independent growth. Phosphorylation of the retinoblastoma (Rb) protein [a target of the cyclin D1/cyclin-dependent kinase 4/6 (cdk4/6) holoenzyme] was delayed in the cyclin D1(-/-) cells relative to cyclin D1(-/+) cells. The E7 oncogene, whose activity includes degradation of Rb and dissociation of Rb from E2F, did not confer anchorage-independent growth to the cyclin D1(-/-) cells but, in conjunction with vSrc, resulted in robust growth in soft agar. These results suggest both a cdk-dependent and cdk-independent role for cyclin D1 in modulating transformation by different oncogenes.

Animals↗
PubMed2005

Reduced STAT3 activity in mice mimics clinical disease syndromes.

Phosphorylation on Y705 is obligatory for STAT3 activation, but full transcriptional activity of this widely expressed protein also requires phosphorylation on S727. We described earlier the STAT3 SA/- mice (SA, S727A allele) on a Black 6 (Bl6) background that showed 75% perinatal lethality and early growth retardation presumably due to the decreased transcription supported by STAT3 S727A. We now report additional analyses of long-term surviving SA/- animals which show no important tissue abnormalities. However, we have found a much greater susceptibility to doxorubicin-induced heart failure in the SA/- mice. Also we introduced the SA allele into strain 129 and found the SA/- mice showed greater susceptibility to LPS-induced toxicity. These results suggest a continued need for normal STAT3 transcriptional activity to resist two different noxious challenges that mimic the conditions necessary to induce adult diseases.

Animals↗
PubMed2005

Structural bases of unphosphorylated STAT1 association and receptor binding.

The crystal structure has been determined at 3.0 A resolution for an unphosphorylated STAT1 (1-683) complexed with a phosphopeptide derived from the alpha chain of interferon gamma (IFNgamma) receptor. Two dimer interfaces are seen, one between the N domains (NDs) (amino acid residues 1-123) and the other between the core fragments (CFs) (residues 132-683). Analyses of the wild-type (wt) and mutant STAT1 proteins by static light scattering, analytical ultracentrifugation, and coimmunoprecipitation suggest that STAT1 is predominantly dimeric prior to activation, and the dimer is mediated by the ND interactions. The connecting region between the ND and the CF is flexible and allows two interconvertable orientations of the CFs, termed "antiparallel" or "parallel," as determined by SH2 domain orientations. Functional implications of these dimer conformations are discussed. Also revealed in this structure is the detailed interaction between STAT1 SH2 domain and its docking site on IFNgamma receptor.

Crystallization↗
PubMed2005

Implications of an antiparallel dimeric structure of nonphosphorylated STAT1 for the activation-inactivation cycle.

IFN-gamma 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:coil/DNA-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.

Biomarkers↗
PubMed2004

Transformation fingerprint: induced STAT3-C, v-Src and Ha-Ras cause small initial changes but similar established profiles in mRNA.

Induced transformation of mouse fibroblasts was carried out by releasing tetracycline-repressed expression of an oncogenic mutant of STAT3, STAT3-C, or of v-Src or Ha-Ras. At 15 days after derepression of each oncogene, DNA microarrays showed elevation (>3-fold) of a similar group of approximately 25 mRNAs compared to untransformed cells. RT-PCR confirmed a number of these mRNA elevations. RNA samples were then analysed at intervals during the first 24 h after doxycycline removal to determine the time of early changes. Extensive changes were not observed by array analysis, except in v-Src-expressing cells where about 10 mRNAs were elevated threefold or more. However, RT-PCR did uncover changes in each derepressed cell type that included some of the changes observed after the 15-day transformation period. In addition, STAT3-C target genes such as BclXI and cyclin D1, which were not observed on array analysis, were elevated by RT-PCR analysis. We conclude, therefore, that early after oncogene induction, transcriptional changes, including those initiated by STAT3-C, may occur only in scarce mRNA and/or to a limited extent. However, with additional time and probably additional cell division, a new epigenetic state is established that is mirrored by a changed transcriptional profile emblematic of transformation by each of three oncogenes.

Animals↗
PubMed2003

STAT3-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the alpha 2-macroglobulin gene.

We describe a detailed time course of the assembly and disassembly of a STAT3-dependent, glucocorticoid-supplemented enhanceosome for the alpha2-macroglobulin (alpha2-M) gene and compare this with a detailed time course of transcription of the gene by run-on analysis. The glucocorticoid receptor (GR) can associate with the enhanceosome without STAT3. Furthermore, the enhanceosome contains c-Jun/c-Fos and OCT-1 constitutively. All of these factors (GR, c-Jun, OCT-1) have transcription activation domains, but STAT3 is required for the observed transcriptional increase. The time course of enhanceosome occupation by GR and tyrosine-phosphorylated STAT3 shows that these transcription factors precede by approximately 5-10 min the arrival of RNA polymerase II (Pol II). The enhanceosome remains assembled for approximately 90 min in the continued presence of both inducers. When IL-6 and Dex are removed (after 30 min of treatment), the disappearance within an additional 30 min of the established enhanceosome indicates that renewal of STAT3 and GR binding must occur in the continued presence of IL-6+Dex. Compared with the total nuclear tyrosine-phosphorylated STAT3 capable of binding DNA, the chromatin-associated STAT3 resists dephosphorylation and appears to recycle to maintain the enhanceosome. Run-on transcription shows a lag after full enhanceosome occupation that can be largely but not completely explained by the approximately 30 min transit time of Pol II across the alpha2-Mlocus.

Animals↗
PubMed2003

Distinct transcriptional activation functions of STAT1alpha and STAT1beta on DNA and chromatin templates.

Interferon-induced transcription depends upon tyrosine phosphorylation, subsequent dimerization, and binding to DNA of STAT1. Other factors, including but not necessarily limited to CBP/p300, then bind within the C-terminal 38 amino acid transactivation domain (TAD) to activate transcription. We show that both tyrosine-phosphorylated STAT1alpha (full-length wild-type protein) and STAT1beta (lacking the TAD) stimulate in vitro transcription on a naked DNA template. Furthermore, in a system with purified proteins and naked DNA, STAT1alpha- and STAT1beta-dependent transcription is stimulated by the TRAP/Mediator co-activator complex. Thus STAT1, through some site other than the C-terminal TAD, can interact with TRAP/Mediator or some intermediate protein. Although both STAT1alpha and STAT1beta bind to known STAT sites within in vitro assembled chromatin templates, only STAT1alpha, and not STAT1beta, in cooperation with p300 and acetyl-CoA, stimulated in vitro transcription from chromatin. After interferon-gamma treatment, cells recruit STAT1alpha or -beta to the chromosomal interferon-1 gene, but only STAT1alpha-containing cells recruit p300 and stimulate transcription. We conclude that chromatin remodeling by p300 in vivo makes TRAP/Mediator effective in stimulating transcription.

Animals↗
PubMed2003

Independent and cooperative activation of chromosomal c-fos promoter by STAT3.

The c-fos gene was one of the earliest vertebrate genes shown to be transcriptionally induced by growth factors. Intensive study of the promoter of c-fos (-325 to -80) by transient or permanent transfections of synthetic DNA constructs has repeatedly shown the importance of several sequence elements and the resident nuclear proteins that bind them (e.g. ternary complex factor/ELK1; serum response factor, cAMP response element-binding protein/amino-terminal fragment/AP-1). However these studies have left unanswered numerous questions about the role of these proteins in the regulation of the native chromosomal gene. In particular, the role of a site in this enhancer that binds STATs has been controversial. We present evidence here that STAT3 and not STAT1 accumulates on the chromosomal c-fos promoter and provides a boost to transcription without the activation of resident nuclear proteins through serine kinases. Also, when resident nuclear proteins such as ELK1 are activated to varying extents by mitogen-activated protein kinase pathways, STAT3 activation provides a 2-fold boost regardless of the final level of activated transcription. Thus the several proteins that interact with the c-fos enhancer apparently can act either in a cooperative or independent manner to achieve very different levels of transcription.

DNA↗
PubMed2003

Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes.

Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species. Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes. The results show statistically significant organizational principles in the variation of decay rates among functional classes. In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in "fast-decaying" mRNAs with half-lives <2 h. In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs. Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes. Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript. Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay. However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites.

Base Composition↗
PubMed2003

A reinterpretation of the dimerization interface of the N-terminal domains of STATs.

The crystal structures of the N-terminal domain (N-domain) and the core region of the STAT family of transcription factors have been determined previously. STATs can form cooperative higher order structures (tetramers or higher oligomers) while bound to DNA. The crystal packing in the STAT4 N-domain crystal structure, determined at 1.5 A resolution, suggests two alternate organizations of the N-domain dimer. We now present the results of site directed mutagenesis of residues predicted to be involved at each dimer interface. Our results indicate that the dimer interface suggested earlier as being physiologically relevant is, in fact, unlikely to be so. Given the alternative model for the N-domain dimer, the ability of the N-domain to mediate interactions of two STAT dimers on DNA remains unchanged.

Binding Sites↗
PubMed2002

Negative regulation of STAT92E by an N-terminally truncated STAT protein derived from an alternative promoter site.

Previously unrecognized mRNAs originating from a dual promoter at the stat92E locus are described. One of these encodes a truncated protein, DeltaNSTAT92E, 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 DeltaNSTAT92E early in embryogenesis. By increasing the DeltaNSTAT92E-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.

Alternative Splicing↗
PubMed2002

Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-gamma.

In response to IFN-gamma, the latent cytoplasmic protein signal transducers and activators of transcription 1 (Stat1) becomes phosphorylated on Y701, dimerizes, and accumulates in the nucleus to activate transcription of IFN-gamma-responsive genes. For maximal gene activation, S727 in the transcription activation domain of Stat1 also is inducibly phosphorylated by IFN-gamma. We previously purified a group of nuclear proteins that interact specifically with the Stat1 transcription activation domain. In this report, we identified one of them as the multifunctional Ca(2+)/calmodulin-dependent kinase (CaMK) II. We demonstrate that IFN-gamma mobilizes a Ca(2+) flux in cells and activates CaMKII. CaMKII can interact directly with Stat1 and phosphorylate Stat1 on S727 in vitro. Inhibition of Ca(2+) flux or CaMKII results in a lack of S727 phosphorylation and Stat1-dependent gene activation, suggesting in vivo phosphorylation of Stat1 S727 by CaMKII. Thus two different cellular signaling events, IFN-gamma receptor occupation and Ca(2+) flux, are required for Stat1 to achieve maximal transcriptional activation through regulation of phosphorylation.

3T3 Cells↗
PubMed2002

Dissociation time from DNA determines transcriptional function in a STAT1 linker mutant.

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 gamma. 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.

Animals↗
PubMed2002

Signal transduction and the control of gene expression.

More than 2000 transcription factors are encoded in the human genome. Such proteins have often been classified according to common structural elements. But because transcription factors evolved in the service of biologic function, we propose an alternative grouping of eukaryotic transcription factors on the basis of characteristics that describe their roles within cellular regulatory circuits.

Animals↗