Quiescent T cells can be induced to express many genes by mitogen or antigen stimulation. The messenger RNAs of some of these genes undergo relatively rapid degradation compared to messenger RNAs from constitutively expressed genes. A T cell activation pathway that specifically regulates the stability of messenger RNAs for the lymphokines interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor is induced by stimulation of the CD28 surface molecule. This pathway does not directly affect the steady-state messenger RNA level, transcription, or messenger RNA half-life of other T cell activation genes, including c-myc, c-fos, IL-2 receptor, and the 4F2HC surface antigen. These data show that stimuli received at the cell surface can alter gene expression by inducing specific changes in messenger RNA degradation.

The dideoxy chain termination method using deoxy-7-deazaguanosine triphosphate (dc7GTP) in place of dGTP was found to be very useful. Sequencing of a part of the human N-myc gene having 85% GC content is impossible by the original method using dGTP, because of compression of bands. However, the nucleotide sequence of this part was unambiguously determined by analysis of both strands by the modified method. Use of dc7GTP is concluded to improve the dideoxy chain termination method for DNA sequencing.

MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

MicroRNAs (miRNAs) are endogenously expressed RNAs, 18-25 nucleotides in length, that repress protein translation through binding to target mRNAs. miRNAs have been implicated in many cellular processes including cell proliferation, differentiation, and death. Recently, let-7 miRNAs were found to regulate human RAS oncogene expression and to be often down-regulated in human lung tumors. In this study, we examined the expression of let-7 miRNAs in human colon cancer tumors and cell lines, with the result that 2 of 6 cases and 1 of 3 cell lines showed reduced expression of let-7. When let-7 low-expressing DLD-1 human colon cancer cells were transfected with let-7a-1 precursor miRNA, which is located at chromosome 9q22.3, the cells underwent significant growth suppression. At that time, the levels of RAS and c-myc proteins were lowered after the transfection, whereas the levels of both of their mRNAs remained almost unchanged. These findings suggest the involvement of let-7 miRNA in the growth of colon cancer cells. Thus, miRNAs might provide a basis for novel RNA anti-cancer agents.

The transcription factor IRF4 (interferon regulatory factor 4) is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Multiple myeloma, a malignancy of plasma cells, has a complex molecular aetiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations. Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses. Current therapies for myeloma can extend survival but are not curative. Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Here we show, using a loss-of-function, RNA-interference-based genetic screen, that IRF4 inhibition is toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Although IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programmes of normal plasma cells and activated B cells.

Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p < 0.0001). No G34V/R-H3.3, IDH1/2 or H3.1 mutations were identified. K27M-H3.3 DIPGs showed specific copy number changes, including all gains/amplifications of PDGFRA and MYC/PVT1 loci. Notably, all long-term survivors were H3.3 wild type and this group of patients had better overall survival. K27M-H3.3 mutation defines clinically and biologically distinct subgroups and is prevalent in DIPG, which will impact future therapeutic trial design. K27M- and G34V-H3.3 have location-based incidence (brainstem/cortex) and potentially play distinct roles in pediatric GBM pathogenesis. K27M-H3.3 is universally associated with short survival in DIPG, while patients wild-type for H3.3 show improved survival. Based on prognostic and therapeutic implications, our findings argue for H3.3-mutation testing at diagnosis, which should be rapidly integrated into the clinical decision-making algorithm, particularly in atypical DIPG.

Although restoration of p53 function is an attractive tumor-specific therapeutic strategy, it remains unclear whether p53 loss is required only for transition through early bottlenecks in tumorigenesis or also for maintenance of established tumors. To explore the efficacy of p53 reinstatement as a tumor therapy, we used a reversibly switchable p53 knockin (KI) mouse model that permits modulation of p53 status from wild-type to knockout, at will. Using the well-characterized Emu-myc lymphoma model, we show that p53 is spontaneously activated when restored in established Emu-myc lymphomas in vivo, triggering rapid apoptosis and conferring a significant increase in survival. Nonetheless, reimposition of p53 function potently selects for emergence of p53-resistant tumors through inactivation of p19(ARF) or p53. Our study provides important insights into the nature and timing of p53-activating signals in established tumors and how resistance to p53 evolves, which will aid in the optimization of p53-based tumor therapies.

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

Proviruses of avian leukosis virus (ALV) are located in the vicinity of a putative cellular oncogene (c-myc) in ALV-induced bursal lymphomas. Enhanced expression of c-myc occurs in association with proviruses found in any of three configurations: (I) on the 5' side ('upstream') of c-myc in the same transcriptional orientation; (II) on the 3' side ('downstream') of c-myc in the same orientation; (III) upstream, in the transcriptional orientation opposite to that of c-myc. Thus, activation of adjacent cellular genes by retroviral DNA can involve mechanisms other than provision of a transcriptional promoter.

We used the interaction trap to isolate a novel human protein that specifically interacts with Max. This protein, Mxi1 (for Max interactor 1), contains a bHLH-Zip motif that is similar to that found in Myc family proteins. Mxi1 interacts specifically with Max to form heterodimers that efficiently bind to the Myc-Max consensus recognition site. When bound to DNA by a LexA moiety in yeast, Mxi1 does not stimulate transcription. mxi1 mRNA is expressed in many tissues, and its expression is elevated in U-937 myeloid leukemia cells that have been stimulated to differentiate. These facts are consistent with a model in which Mxi1-Max heterodimers indirectly inhibit Myc function in two ways: first, by sequestering Max, thus preventing the formation of Myc-Max heterodimers, and second, by competing with Myc-Max heterodimers for binding to target sites.

We have determined the sequence of the normal human c-myc gene and compared it to portions of a c-myc gene that has been translocated into the immunoglobulin heavy chain locus in a Burkitt lymphoma cell. The normal c-myc gene is encoded in three discrete exons divided by two large intervening sequences. Its mRNA is transcribed from two active promoters located about 150 nucleotides from one another. Each promoter initiates transcription of a long (approximately 550 bp) untranslatable leader sequence encoding the entire first exon. This exon and additional 5' flanking sequences are tightly conserved between mouse and man. In the Burkitt cell BL22, the rearranged c-myc gene retains both promoters and is unchanged in its amino acid coding domains. Translocation of this gene joins it to the immunoglobulin heavy chain switch region at a point approximately 1000 bp 5' to the dual c-myc promoters. These genes are joined in opposite transcriptional orientation. The structure of the translocated gene and the nature of its linkage to the immunoglobulin locus and the presence of two c-myc promoters and consequently two long leader sequences raise novel possibilities for the activation of an oncogene.

Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors. Mxi1-mediated inhibition of Myc activities requires interaction with mammalian Sin3A or Sin3B proteins, which have been purported to act as scaffolds for additional co-repressor factors. The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumour suppression.

We have isolated the cDNA encoding a novel human myogenic factor, Myf-5, by weak cross-hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf-5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf-5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 'fibroblasts'. The existence of more than one MyoD1-like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf-5 in 10T1/2 cells leads to the activation of a co-transfected muscle-specific CAT reporter gene which by itself is transcriptionally silent in the non-muscle cell background. The deduced amino acid sequence of clone Myf-5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Homozygosity for the G allele of rs6983267 at 8q24 increases colorectal cancer (CRC) risk approximately 1.5 fold. We report here that the risk allele G shows copy number increase during CRC development. Our computer algorithm, Enhancer Element Locator (EEL), identified an enhancer element that contains rs6983267. The element drove expression of a reporter gene in a pattern that is consistent with regulation by the key CRC pathway Wnt. rs6983267 affects a binding site for the Wnt-regulated transcription factor TCF4, with the risk allele G showing stronger binding in vitro and in vivo. Genome-wide ChIP assay revealed the element as the strongest TCF4 binding site within 1 Mb of MYC. An unambiguous correlation between rs6983267 genotype and MYC expression was not detected, and additional work is required to scrutinize all possible targets of the enhancer. Our work provides evidence that the common CRC predisposition associated with 8q24 arises from enhanced responsiveness to Wnt signaling.

BACKGROUND

The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma.

METHODS

Tumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation.

RESULTS

A classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappaB target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens.

CONCLUSIONS

Gene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma.

Since its discovery as a protein associated with the cytoplasmic region of E-cadherin, beta-catenin has been shown to perform two apparently unrelated functions: it has a crucial role in cell-cell adhesion in addition to a signaling role as a component of the Wnt/wg pathway. Wnt/wg signaling results in beta-catenin accumulation and transcriptional activation of specific target genes during development. It is now apparent that deregulation of beta-catenin signaling is an important event in the genesis of a number of malignancies, such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer. beta-catenin mutations appear to be a crucial step in the progression of a subset of these cancers, suggesting an important role in the control of cellular proliferation or cell death. The APC/beta-catenin pathway is highly regulated and includes players such as GSK3-beta, CBP, Groucho, Axin, Conductin, and TCF. c-MYC and cyclin D1 were recently identified as a key transcriptional targets of this pathway and additional targets are likely to emerge. Published 1999 John Wiley & Sons, Inc.

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.

The miR-17 approximately 92 cluster is frequently amplified or overexpressed in human cancers and has emerged as the prototypical oncogenic polycistron microRNA (miRNA). miR-17 approximately 92 is a direct transcriptional target of c-Myc, and experiments in a mouse model of B-cell lymphomas have shown cooperation between these two oncogenes. However, both the molecular mechanism underlying this cooperation and the individual miRNAs that are responsible for it are unknown. By using a conditional knockout allele of miR-17 approximately 92, we show here that sustained expression of endogenous miR-17 approximately 92 is required to suppress apoptosis in Myc-driven B-cell lymphomas. Furthermore, we show that among the six miRNAs that are encoded by miR-17 approximately 92, miR-19a and miR-19b are absolutely required and largely sufficient to recapitulate the oncogenic properties of the entire cluster. Finally, by combining computational target prediction, gene expression profiling, and an in vitro screening strategy, we identify a subset of miR-19 targets that mediate its prosurvival activity.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1alpha, a basic helix-loop-helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1alpha for hypoxia-induced growth arrest and activation of p21(cip1), a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21(cip1) activation has been elusive. Here we demonstrate that HIF-1alpha, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21(cip1). The HIF-1alpha antagonism is mediated by displacing Myc binding from p21(cip1) promoter. Neither HIF-1alpha transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1alpha. In keeping with its antagonism of Myc, HIF-1alpha also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1alpha pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. We used chromatin immunoprecipitation (ChIP) to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc associated with chromatin, histone H4 became locally hyperacetylated, and gene expression was induced. These responses were lost or severely impaired in Myc-deficient cells, but were restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induced H4 acetylation. In addition, Myc recruited TRRAP to chromatin, consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone was very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. We propose that Myc acts as a permissive factor, allowing additional signals to activate target promoters.

We identified a gene of the fungal pathogen Candida albicans, designated EFG1, whose high-level expression stimulates pseudohyphal morphogenesis in the yeast Saccharomyces cerevisiae. In a central region the deduced Efg1 protein is highly homologous to the StuA and Phd1/Sok2 proteins that regulate morphogenesis of Aspergillus nidulans and S. cerevisiae, respectively. The core of the conserved region is homologous to the basic helix-loop-helix (bHLH) motif of eukaryotic transcription factors, specifically to the human Myc and Max proteins. Fungal-specific residues in the bHLH domain include the substitution of an invariant glutamate, responsible for target (E-box) specificity, by a threonine residue. During hyphal induction EFG1 transcript levels decline to low levels; downregulation is effected at the level of transcriptional initiation as shown by a EFG1 promoter-LAC4 fusion. A strain carrying one disrupted EFG1 allele and one EFG1 allele under the control of the glucose-repressible PCK1 promoter forms rod-like, pseudohyphal cells, but is unable to form true hyphae on glucose-containing media. Overexpression of EFG1 in C. albicans leads to enhanced filamentous growth in the form of extended pseudohyphae in liquid and on solid media. The results suggest that Efg1p has a dual role as a transcriptional activator and repressor, whose balanced activity is essential for yeast, pseudohyphal and hyphal morphogenesis of C. albicans. Functional analogies between Efg1p and Myc are discussed.