Judith Chebath

Interferon-activated genes

Abstract Interferons (IFNs) are cell-secreted protein factors which are not only involved in the defence against virus infections, but play a key role in the regulation of cell growth and differentiation. More than 10 cellular genes have now been identified as being specifically induced by IFNs and hence possible mediators of the complex biological action of IFNs. Transcription of several of these genes is activated within minutes following interaction of IFNs with their cell surface receptors.

A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor

The intracytoplasmic domain (IC) of cytokine receptors provides docking sites for proteins which mediate signal transduction. Thus, in interferon‐α,β receptors (IFNAR1 and 2), the IC region binds protein‐tyrosine and ‐serine/threonine kinases which phosphorylate the receptor and the associated Stat transcription factors. A two‐hybrid screening was carried out to identify additional proteins which could interact with the IC domain of the IFNAR1 chain of the IFN‐α,β receptor. Several positive clones representing a protein sequence designated IR1B4 were recovered from a human cDNA library. IR1B4 was identified as the human homolog of PRMT1, a protein‐arginine methyltransferase from rat cells. Flag‐IR1B4 fusion proteins bind to the isolated IFNAR1 intracytoplasmic domain produced in Escherichia coli, as well as to the intact IFNAR1 chain extracted by detergent from human U266 cell membranes. S‐Adenosylmethionine‐dependent methyltransferase activity was precipitated by anti‐IFNAR1 antibodies from untreated human cells. IR1B4/PRMT1 is involved in IFN action since U266 cells rendered deficient in this methyltransferase by antisense oligonucleotides become more resistant to growth inhibition by IFN. Methylation of proteins by enzymes which can attach to the IC domains of receptors may be a signaling mechanism complementing protein phosphorylation. Among substrates methylated by PRMT1 are RNA‐binding heterogeneous nuclear ribonucleoproteins (hnRNPs) which are involved in mRNA processing, splicing and transport into the cytoplasm.

Structure of two forms of the interferon-induced (2'-5') oligo A synthetase of human cells based on cDNAs and gene sequences.

The (2′‐5′) oligo A synthetase E, one of the translational inhibitory enzymes whose synthesis is strongly induced by all interferons (IFNs), is shown to be encoded in human cells by a 13.5‐kb gene. By a cell‐specific differential splicing, between the seventh and an additional eighth exon of this gene, two active E mRNAs of 1.6 and 1.8 kb are produced, along with several longer transcripts. cDNA clones for the two mRNAs were obtained and their sequences indicate that the human (2′‐5′) oligo A synthetase gene codes for two forms of the enzyme of mol. wt. 41 000 and 46 000, which differ only by their C‐terminal ends. The product of the 1.6‐kb RNA (E16) has a very hydrophobic C terminus, which is replaced by a longer acidic C‐terminal sequence in the 1.8‐kb RNA product (E18). The transcriptional start site of the gene was identified and 200 bp of the 5′ flanking region were sequenced. A strong homology was found between this region of the IFN‐activated (2′‐5′) oligo A synthetase gene and the corresponding region of the human fibroblast IFN‐beta 1 gene, whose transcription is also stimulated by IFN priming. The gene has two polyadenylation sites which share a common undecanucleotide, but are used in a cell‐specific manner to give rise to the 1.6‐ and 1.8‐kb mRNAs.

Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo.

In attempts to produce mature oligodendrocytes from human embryonic stem (huES) cells, we searched conditions inducing transcription factors Olig1/2, as well as Nkx2.2 and Sox10, which are needed for maturation. This was obtained by retinoic acid treatment followed by noggin, an antagonist of bone morphogenetic proteins (BMPs). We found that retinoic acid induces BMPs in huES cells. Addition of noggin at a specific step was essential to form numerous mature oligodendrocytes with ramified branches and producing myelin basic protein (MBP). We describe a procedure converting huES cells into enriched populations of oligodendrocyte precursors that can be expanded and passaged repeatedly and subsequently differentiated into mature cells. Transplantation of such precursors showed that pretreatment by noggin markedly stimulates their capacity to myelinate in the brain of MBP-deficient shiverer mice in organotypic cultures and in living animals. Arrays of numerous long MBP+ fibers were generated over extended areas in the brain, with evidence of cell migration after transplantation and with formation of compact myelin sheaths.

Enhancer-like interferon responsive sequences of the human and murine (2'-5') oligoadenylate synthetase gene promoters.

The human (2′‐5′) oligo(A) synthetase gene contains two independent cis‐acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN‐inducibility to the herpes tk promoter. Two murine (2′‐5′) oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK‐CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon‐responsive sequence (E‐IRS) were cloned in 5′ of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E‐IRS. This binding is competed by the addition of cold synthetic mouse E‐IRS or fragments of genomic DNA containing the E‐IRS.

Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells

Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.

Induction by interleukin-6 of interferon regulatory factor 1 (IRF-1) gene expression through the palindromic interferon response element pIRE and cell type-dependent control of IRF-1 binding to DNA.

The effects of interleukin‐6 (IL‐6) on interferon regulatory factor 1 (IRF‐1) gene expression were studied in B‐hybridoma B9 cells which are growth‐stimulated by IL‐6 and breast carcinoma T47D cells which are growth‐inhibited. IL‐6 induced the production of IRF‐1 mRNA and protein in both cell types, but IRF‐1 binding activity to its target DNA sequence was induced only in T47D cells. With B9 cells, there was no IRF‐1 binding but instead strong constitutive binding of the IRF‐2 repressor, indicating that binding of IRF‐1 to DNA is an important regulatory step. The IRF‐1 gene promoter element, palindromic IFN‐response element (pIRE), was found to respond to IL‐6 with high efficiency as compared with IFN‐gamma or IFN‐beta. On this palindromic TTC…GAA sequence, two protein complexes (pIRE‐a and pIRE‐b) were induced within minutes by IL‐6. pIRE‐b is similar to the main complex induced by IFN‐gamma and contains the Stat91 protein. pIRE‐a predominantly induced by IL‐6 is a slowly migrating complex which does not contain Stat91 and has low affinity for IFN‐gamma activated sequence (GAS)‐type sequences. Comparison of the relative effects of IL‐6 and IFN‐gamma shows that pIRE enhancers are differently regulated than GAS elements. Distinct transcription complexes, forming in ratios dependent on the inducer, help explain how various cytokines sharing effects through Stat91 on related enhancers can produce specific patterns of gene expression. Activation of the pIRE‐a factors defines a novel transcriptional activity of IL‐6 in epithelial and lymphoid cells.

Pax3 down-regulation and shut-off of melanogenesis in melanoma B16/F10.9 by interleukin-6 receptor signaling

The microphthalmia-associated transcription factor (Mitf) is essential for melanocytic lineage development and for expression of melanogenic enzymes, such as tyrosinase. Interleukin-6 receptor/interleukin-6 chimera (IL6RIL6) induces in B16/F10.9 melanoma cells a loss of melanogenesis preceded by a sharp decrease in Mitf mRNA and gene promoter activity. In the Mitf promoter, the main cis-acting element mediating the IL6RIL6 effect is shown to be the binding site of Pax3, a paired homeodomain factor regulating among other things the development of melanocytes. Pax3 protein and mRNA levels decline steadily after IL6RIL6 treatment, and overexpression of an ectopic Pax3 cDNA suppresses the Mitf promoter inhibition. Loss of the synergism between Pax3 and Sox10, a high mobility group domain costimulatory factor, seems to be critical in the rapid decrease in Mitf gene expression. The Pax3 down-regulation in IL6RIL6-induced F10.9 cell is linked to growth arrest and transdifferentiation to a glial cell phenotype. IL6RIL6 stimulates the interleukin-6 family cytokine receptor gp130, leading to the rapid phosphorylation of Stat3 on tyrosine 705. This phosphorylation is required for Pax3 down-regulation and Mitf promoter silencing since these are inhibited in F10.9 cells overexpressing the Stat3 DN-mutant Y705F.

Involvement of receptor-bound protein methyltransferase PRMT1 in antiviral and antiproliferative effects of type I interferons.

Protein arginine N-methyltransferase (PRMT1) is one of the proteins that bind to the intracytoplasmatic domain of the IFNAR-1 chain of the type I interferon (IFN) receptor system. The attachment is specific and is not seen with PRMT2, another member of this protein family. Antisense PRMT1 cDNA constructs expressed under the early cytomegalovirus (CMV) promoter were transfected into HeLa cells, and stable transformants were selected. Antibodies to PRMT1 were used to identify clones with reduced PRMT1 expression. In such clones, IFN-beta inhibited three to five times less the replication of vesicular stomatitis virus (VSV) than in the original HeLa cells. The antiproliferative effect of IFN-beta was also reduced up to fivefold in the clones with low PRMT1 expression. No difference was seen when IFN-gamma was used alone to inhibit cell growth. The protein methylating enzyme, bound to IFNAR-1, appears to regulate positively the biologic activity of type I IFN.

Increased myelinating capacity of embryonic stem cell derived oligodendrocyte precursors after treatment by interleukin-6/soluble interleukin-6 receptor fusion protein

Neurosphere cells (NSc) derived from embryonic stem cells have characteristics of neural stem cells and can differentiate into oligodendrocyte precursors. Culture of NSc with IL6RIL6 chimera (soluble interleukin-6 receptor fused to interleukin-6) enhances their differentiation into oligodendrocytes with longer and more numerous branches and with peripheral accumulation of myelin basic protein (MBP) in myelin membranes indicating maturation. Gene expression profiling reveals that one of the proteins strongly induced by IL6RIL6 is a regulator of microtubule dynamics, stathmin-like 2 (SCG10/Stmn2), and gene silencing shows that Stmn2 plays an important role in the development of the mature oligodendrocyte morphology. IL6RIL6 acts as an effective stimulator of the myelinating function of ES cell-derived oligodendrocyte precursors, as observed upon transplantation of the IL6RIL6- pretreated cells into brain slices of MBP-deficient shiverer mice.