Moitreyee Chatterjee-Kishore

How Stat1 mediates constitutive gene expression: a complex of unphosphorylated Stat1 and IRF1 supports transcription of the LMP2 gene

Analysis of mRNA levels in cells that express or lack signal transducers and activators of transcription 1 (Stat1) reveals that Stat1 mediates the constitutive transcription of many genes. Expression of the low molecular mass polypeptide 2 (LMP2), which requires Stat1, has been studied in detail. The overlapping interferon consensus sequence 2/γ‐interferon‐activated sequence (ICS‐2/GAS) elements in the LMP2 promoter bind to interferon regulatory factor 1 (IRF1) and Stat1 and are occupied constitutively in vivo. The point mutant of Stat1, Y701F, which does not form dimers involving SH2–phosphotyrosine interactions, binds to the GAS element and supports LMP2 expression. Unphosphorylated Stat1 binds to IRF1 directly and we conclude that this complex uses the ICS‐2/GAS element to mediate constitutive LMP2 transcription in vivo. The promoter of the IRF1 gene, which also contains a GAS site but not an adjacent ICS‐2 site, is not activated by Stat1 Y701F. The promoters of other genes whose constitutive expression requires Stat1 may also utilize complexes of unphosphorylated Stat1 with IRF1 or other transcription factors.

Complex roles of Stat1 in regulating gene expression

Stat1 is a fascinating and complex protein with multiple, yet contrasting transcriptional functions. Upon activation, it drives the expression of many genes but also suppresses the transcription of others. These opposing characteristics also apply to its role in facilitating crosstalk between signal transduction pathways, as it participates in both synergistic activation and inhibition of gene expression. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes. This review summarizes the well studied involvement of Stat1 in IFN-dependent and growth factor-dependent signaling and then describes the roles of Stat1 in positive, negative and constitutive regulation of gene expression as well as its participation in crosstalk between signal transduction pathways.

Association of STATs with relatives and friends.

Members of the STAT family of transcription factors are present in species as diverse as mammals, insects and slime molds. Discovered as mediators of interferon-induced signals, the STATs were later shown to drive many different ligand-induced responses through receptor-induced tyrosine phosphorylation and dimerization. STAT1 also functions as a transcription factor, essential for the efficient constitutive expression of certain genes, without needing tyrosine phosphorylation, and phosphorylated STAT1 dimers mediate suppression - rather than activation - of some genes. STATs are present in the cytoplasm of untreated cells in multiprotein complexes, which might aid in their nuclear translocation and differential binding to DNA, thus contributing to the specificity of STAT action. This review explores the diverse protein-protein interactions that underlie the multiple functions of the STATs.

Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

ABSTRACT 7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21waf1, or Stat1, we found that optimal 7kchol-induced apoptosis requires p21waf1 and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser727 but not Tyr701 was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-β) blunted Ser727 phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-β receptor subunit exhibited blunted apoptosis. IFN-β alone did not induce apoptosis; thus, 7kchol-induced release of IFN-β was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21waf1 was much less than in wild-type cells; introducing transient expression of p21waf1 restored apoptosis. Stat1 and p21waf1 were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.

Adenovirus E1A Down-regulates LMP2 Transcription by Interfering with the Binding of Stat1 to IRF1

The LMP2 gene, which encodes a protein required for efficient presentation of viral antigens, requires both unphosphorylated Stat1 and IRF1 for basal expression. LMP2 expression is down-regulated by the adenovirus protein E1A, which binds to Stat1 and CBP/p300, and by the mutant E1A protein RG2, which binds to Stat1 but not to CBP/p300, but not by the mutant protein Δ2–36, which does not bind to either Stat1 or CBP/p300. Stat1 and IRF1 associate in untreated cells and bind as a complex to the overlapping ICS-2/GAS element of the LMP2 promoter. E1A interferes with the formation of this complex by occupying domains of Stat1 that bind to IRF1. These results reveal how adenovirus infection attenuates LMP2 expression, thereby interfering with the presentation of viral antigens.

Resistance to Interferons in Melanoma Cells Does Not Correlate with the Expression or Activation of Signal Transducer and Activator of Transcription 1 (Stat1)

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

STAT-Dependent Gene Expression without Tyrosine Phosphorylation

The initial discovery that Stats 1 and 2 are activated in response to IFNs through the phosphorylation of specific tyrosine residues, followed by dimerization through phosphotyrosine-SH2 interactions (Figure 1A) (1) led quickly to an appreciation that the same mechanism held true for ligand-dependent activation of all of the other Stats. However, more recently, it has been found that Stats 1 and 3 (and probably other Stats) also play important roles in mediating gene expression without tyrosine phosphorylation. When expressed at normal levels, Stats 1 and 3 support the ligand-independent constitutive transcription of certain genes, and abnormally high levels of Stat3, found in many tumors, drive the overexpression of these genes. Furthermore, ligand-dependent transcription, resulting from the phosphorylation on a specific serine residue in the transactivation domain of Stat 1 or 3, can also drive gene expression without tyrosine phosphorylation. Stat1 mediates constitutive expression of the LMP2 gene (Figure 1B) by collaborating with IRF1 (2), and this mechanism is likely to serve as a paradigm in general for how Stats mediate gene expression without tyrosine phosphorylation. Stats are sticky and can interact with many different proteins. Stat1 and IRF1 bind to each other even in the absence of DNA, and this heterodimer binds to a composite element in the LMP2 promoter that recognizes each monomeric component separately. The ternary complex is stable enough to drive the constitutive expression of LMP2 but can be displaced by the more potent Stat1 dimer in IFNγ-treated cells.