Faculty Bibliography

STAT transcription factors have been implicated in many biological processes, particularly host immune defense and development. Here we characterize a STAT orthologue from the nematode, C. elegans. We show that this protein, termed STA-1, is structurally and functionally related to other vertebrate and invertebrate STAT proteins, recognizing a cis DNA element conserved through phylogeny. Unexpectedly, STA-1 lacks the conserved amino-terminal oligomerization domain found in vertebrate and other invertebrate STAT proteins, a feature also lacking in orthologues from a distantly related nematode species and from the slime mold, Dictyostelium discoideum. This absence suggests that a primordial STAT protein lacked this domain, which was accreted later in evolution to provide further regulatory control of STAT signaling. Derivation of null mutants demonstrated that STA-1 is not required for nematode viability, despite its widespread expression in multiple tissues of the worm. However, mutant STA-1 proteins that lack functional coiled-coil and DNA binding domains could still be activated and accumulated in the nucleus, suggesting that DNA binding is not a necessary prerequisite for nuclear retention of activated STAT proteins. Our results shed new light on the evolution and function of the STAT signaling pathway and on the structural requirements for STAT activation

The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences

Measurement of the steady-state abundance of nuclear and cytoplasmic RNA requires efficient subcellular fractionation and RNA recovery coupled with accurate quantification of individual RNA species. Detergent lysis of tissue culture cells provides a simple fractionation procedure that can be optimized to individual cell lines. The large dynamic range, extreme sensitivity, high sequence-specificity, and fast turn-around time has allowed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to become a standard tool for mRNA quantification. Among the different chemistries used for PCR product detection during amplification, DNA binding dyes such as SYBR Green I are simple, versatile, and yet highly reliable and least expensive. With attention to primer design and cycling conditions, virtually any mRNA species can be accurately quantified from even minute quantities of starting RNA. This method provides an accurate and efficient procedure for estimating the relative ratios of nuclear and cytoplasmic RNA concentrations

The tumor suppressor STAT1 is considered a key regulator of the surveillance of developing tumors. Here, we describe an unexpected tumor-promoting role for STAT1 in leukemia. STAT1(-/-) mice are partially protected from leukemia development, and STAT1(-/-) tumor cells induce leukemia in RAG2(-/-) and immunocompetent mice with increased latency. The low MHC class I protein levels of STAT1(-/-) tumor cells enable efficient NK cell lysis and account for the enhanced tumor clearance. Strikingly, STAT1(-/-) tumor cells acquire increased MHC class I expression upon leukemia progression. These findings define STAT1 as a tumor promoter in leukemia development. Furthermore, we describe the upregulation of MHC class I expression as a general mechanism that allows for the escape of hematopoietic malignancies from immune surveillance

Interferon regulatory factor 7 (IRF7) is a key component of the cellular response to virus infection that culminates in physiologically relevant IFNalpha production. We studied molecular mechanisms governing responses to respiratory viral infection that are characterized by transient induction and subsequent shut-off of interferon (IFN) gene expression. We asked whether alterations in IRF7 protein stability occurred during virus infection that might contribute to this regulation. To this end, we measured IRF7 half-life in various cell types and found it to be short-lived, in marked contrast to the pronounced stability of the related transcription factor, IRF3. Furthermore, virus infection accelerated IRF7 degradation in a proteosome-dependent manner in most cell types. However, plasmacytoid dendritic cells (pDC), which constitute the major circulating IFN producing cell type, displayed a distinct pattern of regulation. Infection of lymphoid tissues, where the majority of IRF7 is expressed in pDC, attenuated the normal proteosome-mediated degradation of IRF7, resulting in a long-lived protein. Stabilization was partially stimulated by autocrine IFN as a positive feedback mechanism, but was partially IFN independent. Thus, two distinct posttranslational mechanisms regulate IRF7 activity in response to viral infection, with protein turnover attenuating responses postinfection in most cell types while infection-induced protein stabilization contributes to the heightened IFN production characteristic of pDC

Nup98 and Nup96 are components of the nuclear transport machinery and are induced by interferons (IFN). Nup98 is a constituent of an mRNA export pathway that is targeted by viruses and regulated by IFN. However, the role of Nup96 in IFN-related mechanisms has not been established. To investigate the function of Nup96 in vivo, we generated Nup96(+/-) mice that express low levels of Nup96, as Nup96(-/-) mice are lethal. The Nup96(+/-) mice presented selective alterations of the immune system, which resulted in downregulation and impaired IFN alpha- and gamma-mediated induction of MHC I and IFNgamma induction of MHC II, ICAM-1, and other proteins. Frequency of TCRalphabeta+ and CD4+ T cells, which depends on MHC function, is reduced in NUP96(+/-) mice. Upon immunization, Nup96(+/-) mice showed impaired antigen presentation and T cell proliferation. Nup96(+/-) cells and mice were highly susceptible to viral infection, demonstrating a role for Nup96 in innate and adaptive immunity

The accumulation of myeloid suppressor cells (MSCs) is associated with immune suppression in tumor-bearing mice and in cancer patients. The suppressive activity of MSC correlates with the expression of the myeloid markers Gr-1, CD115 (macrophage colony-stimulating factor receptor), and F4/80. Gr-1(+)CD115(+) MSCs, in addition to being able to suppress T-cell proliferation in vitro, can induce the development of Foxp3(+) T regulatory cells (Treg) in vivo, which are anergic and suppressive. Furthermore, the secretion of interleukin (IL)-10 and transforming growth factor-beta by Gr-1(+)CD115(+) MSCs was induced and enhanced, respectively, on IFN-gamma stimulation. The development of Treg requires antigen-associated activation of tumor-specific T cells, depends on the presence of IFN-gamma and IL-10, and is independent of the nitric oxide-mediated suppressive mechanism by MSC. Our data provide evidence that Gr-1(+)CD115(+) MSC can mediate the development of Treg in tumor-bearing mice and show a novel immune suppressive mechanism by which MSCs can suppress antitumor responses