Interferon-gamma (IFN-gamma) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN-gamma ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN-gamma signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide are discussed.

https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.0603252

Interferon-γ: an overview of signals, mechanisms and functions

MicroRNAs (miRNAs) are 21–23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.

c-MYC-regulated micro RNAs modulate E2F1 expression

MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.

c-Myc-regulated microRNAs modulate E2F1 expression.

MYC on the Path to Cancer

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

Direct observation of individual endogenous protein complexes in situ by proximity ligation

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

https://www.pnas.org/content/pnas/97/5/2229.full.pdf

Identification of CDK4 as a target of c-MYC.

Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +1+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-+S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.

http://cgd.aacrjournals.org/cgi/reprint/8/10/1039.pdf

Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination

c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc-/- cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin-cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27(KIP1) and decreased expression of Cdk7 in c-myc-/- cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions.

/pdf/c-myc-regulates-cyclin-d-cdk4-and-cdk6-activity-but-affects-2niy7buxnc.pdf

c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points.

A large body of physiological evidence shows that either upregulation or downregulation of intracellular c-Myc activity has profound consequences on cell cycle progression. Recent work suggests that c-Myc may stimulate the activity of cyclin E/cyclin-dependent kinase 2 (Cdk2) complexes and antagonize the action of the Cdk inhibitor p27KIP1. Cyclin D/Cdk4/6 complexes have also been implicated as targets of c-Myc activity. However, in spite of considerable effort, the mechanisms by which c-Myc interacts with the intrinsic cyclin/Cdk cell cycle machinery remain undefined.

Mysterious liaisons: the relationship between c-Myc and the cell cycle.

Mutations that disrupt the regulation or expression level of the c-myc gene are frequently found in human and animal cancers (for review, see Henriksson and Luscher 1996). In addition to having a role in uncontrolled cell growth, many observations indicate that c-myc also regulates normal cell proliferation. First, c-myc is expressed in all dividing cells, and, conversely, expression is suppressed once cells withdraw from the cell cycle or terminally differentiate (Marcu et al. 1992). Second, antisense-mediated reduction of c-Myc suggests that it is required for efficient progression through the cell cycle (Yokoyama and Imamoto 1987). Third, ectopic expression of c-Myc or activation of a c-Myc–estrogen receptor fusion protein (Myc–ER) can in some circumstances induce quiescent cells to enter S phase (Armelin et al. 1984; Eilers et al. 1989). Despite substantial effort, the molecular mechanisms by which c-myc controls proliferation and tumorigenesis are not understood.

The c-Myc protein dimerizes with Max and recognizes the c-Myc/Max E–box consensus site (CACGTG) (Henriksson and Luscher 1996). Because transfection of c-myc can transactivate promoters containing this binding site (Kretzner et al. 1992), it is generally believed that c-Myc functions by activating the expression of specific target genes. However, the biggest impediment to understanding c-Myc function is the lack of a comprehensive set of c-Myc target genes. Numerous studies have attempted to identify c-myc targets, mainly using subtractive hybridization, differential display, or educated guessing. The list of candidate transactivation targets includes ornithine decarboxylase (ODC), α-prothymosin, lactate dehydrogenase-A (LDH-A), p53, ECA39, eIF4E, cad, MrDb, rcl, RCC1, and cdc25A (Eilers et al. 1991; Benvenisty et al. 1992; Bello-Fernandez et al. 1993; Reisman et al. 1993; Wagner et al. 1993; Miltenberger et al. 1995; Galaktionov et al. 1996; Grandori et al. 1996; Jones et al. 1996; Lewis et al. 1997; Shim et al. 1997; Tsuneoka et al. 1997). All of the data supporting these genes as bona fide c-Myc targets come from transient or overexpression experiments; consequently, it is not clear whether any of the proposed target genes are necessary for the ability of c-Myc to control cell proliferation.

Although c-Myc clearly is instrumental in directing proliferation, recent studies indicate that it is not absolutely essential for cell growth. Cell lines were derived by targeted homologous recombination in a nontransformed Rat1 fibroblast line to knock out all c-myc expression, and these lines have no detectable N-myc or L-myc expression that might compensate for the absence of c-myc (Mateyak et al. 1997). Although the c-myc null cells continue to grow, they have a dramatically extended cell-cycle time that is nearly three times longer than their wild-type parent. The c-myc null cells have a prolonged G1 and G2 phase, arguing that c-myc functions in both of these phases of the cell cycle (Mateyak et al. 1997). Interestingly, S phase is not prolonged, suggesting that DNA replication is unaltered by c-myc knockout. These c-myc null cell lines provide a powerful new tool for the evaluation of potential c-Myc target genes. In this report we present an analysis of the proposed c-Myc target genes using this novel loss-of-function approach. The results suggest a significant re-evaluation of the proposed collection of c-Myc target genes and indicate that the transactivation of most known cellular targets is not linked to the ability of c-Myc to control the cell cycle.

/pdf/c-myc-null-cells-misregulate-cad-and-gadd45-but-not-other-4n6kyzdxmv.pdf

Alvaro J. Obaya

Papers

Identification of CDK4 as a target of c-MYC.

Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination

c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points.

Mysterious liaisons: the relationship between c-Myc and the cell cycle.

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets