Jeffry R. Cook

Identification and sequence of an accessory factor required for activation of the human interferon γ receptor

Abstract Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon γ (Hu-IFN-γ), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-γ receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-γ receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-γ receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-γ.

Negative regulation of transcription by the type II arginine methyltransferase PRMT5

We have identified previously a repressor element in the transcription start site region of the cyclin E1 promoter that periodically associates with an atypical, high molecular weight E2F complex, termed CERC. Purification of native CERC reveals the presence of the type II arginine methyltransferase PRMT5, which can mono‐ or symetrically dimethylate arginine residues in proteins. Chromatin immunoprecipitations (ChIPs) show that PRMT5 is associated specifically with the transcription start site region of the cyclin E1 promoter. ChIP analyses also show that this correlates with the presence on the same promoter region of arginine‐methylated proteins including histone H4, an in vitro substrate of PRMT5. Consistent with its presence within the repressor complex, forced expression of PRMT5 negatively affects cyclin E1 promoter activity and cellular proliferation, effects that require its methyltransferase activity. These data provide the first direct experimental evidence that a type II arginine methylase is involved in the control of transcription and proliferation.

Interaction between the components of the interferon gamma receptor complex

Interferon γ (IFN-γ) signals through a multimeric receptor complex consisting of two different chains: the IFN-γ receptor binding subunit (IFN-γR, IFN-γR1), and a transmembrane accessory factor (AF-1, IFN-γR2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-γ receptor complex, we examined the function of the receptor components in signal transduction upon IFN-γ treatment. A specific IFN-γR2:IFN-γ cross-linked complex was observed in cells expressing both IFN-γR1 and IFN-γR2 indicating that IFN-γR2 (AF-1) interacts with IFN-γ and is closely associated with IFN-γR1. We show that the intracellular domain of IFN-γR2 is necessary for signaling. Cells coexpressing IFN-γR1 and truncated IFN-γR2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-γR1 alone were unresponsive to IFN-γ treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1α were activated, and IFN-γR1 was phosphorylated only in cells expressing both IFN-γR1 and IFN-γR2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-γR2.

Differential Responsiveness of a Splice Variant of the Human Type I Interferon Receptor to Interferons

Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (αYAC) respond to all type I human interferons including IFN-αA, IFN-β, and IFN-ω. The αYAC contains at least two genes encoding interferon-α receptor (IFN-αR) chains that are required for response to type I human interferons: Hu-IFN-αR1 and Hu-IFN-αR2. We previously isolated a splice variant of the Hu-IFN-αR1 chain designated Hu-IFN-αR1s. Chinese hamster ovary cells containing a disrupted αYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-αR1 and Hu-IFN-αR1s chains. With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-αR1s) and with the Hu-IFN-αR1 chains: Hu-IFN-αA and IFN-ω. Two other type I interferons, Hu-IFN-αB2 and Hu-IFN-αF, are capable of signaling through the Hu-IFN-αR1 chain only and cannot utilize the splice variant Hu-IFN-αR1s. Hu-IFN-αR1 and Hu-IFN-αR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene. These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-αR1 receptor chain, and that the splice variant chain (Hu-IFN-αR1s) is functional.

New vectors for manipulation and selection of functional yeast artificial chromosomes (YACs) containing human DNA inserts

A set of fragmentation vectors is described which produce a deletion series of smaller yeast artificial chromosomes (YACs) from a larger parent YAC with the insertion of a eukaryotic selectable marker. In addition, new vectors were designed to permit integration of the genes encoding neomycin (neo) or hygromycin B (hyg) resistance into YACs containing inserts of human DNA. All these vectors are compatible with the yeast host strain AB1380, in which most human genomic YAC libraries are maintained. Linearized vector DNA is used to transform yeast cells in which homologous recombination between human DNA in the YAC and the Alu sequence in the fragmentation or integrating vector produces terminal deletions from the acentromeric (URA3) end of the YAC or insertion of the vector into the YAC, respectively. A set of directional deletions of a YAC is useful for genomic mapping, restriction analysis and functional measurements of large chromosomal regions. The neo and hyg eukaryotic markers permit the study of gene function after introduction of deleted YACs into mammalian cells. Transformation of YACs with the fragmentation vectors resulted in fragmentation in 21-46% of the clones examined; transformation with the integrating vector resulted in integration in 46% of the clones examined.

Mouse Macrophages Carrying Both Subunits of the Human Interferon-γ (IFN-γ) Receptor Respond to Human IFN-γ but Do Not Acquire Full Protection against Viral Cytopathic Effect

Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-γ receptor (hu-IFN-γR) requires the formation of a complex consisting of ligand (IFN-γ), a ligand binding receptor chain (IFN-γR1), and a signal transducing receptor chain (IFN-γR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-γ can exert on fibroblasts. The current report assesses the ability of hu-IFN-γR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-γR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-γR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-γ with an affinity comparable to that of human cells. Binding of either human or mouse IFN-γ to its respective receptor elicits classic IFN-γ responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2− radicals, interleukin-6, tumor necrosis factor-α, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-γ could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-γ could. These results demonstrate that while co-expression of hu-IFN-γR1 and hu-IFN-γR2 is necessary and sufficient for most IFN-γ-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-γ receptor complex. The nature and potential role of such factors in IFN-γR signaling is discussed.

Yeast artificial chromosome fragmentation vectors that utilize URA3 selection

Two fragmentation vectors, pSE1 and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura- yeast cells containing YACs were selected with 5-fluoro-orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura+ phenotype. Over 80% of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1380 and other ura3- yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells.