c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc-oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain- dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc Box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces α-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated α-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation.

The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. Here we demonstrate that MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts led to rapid cellular senescence that could not be suppressed by hTERT expression. We propose that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence.Supplemental material is available at http://parma.fhcrc.org/ CGrandori. Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma (Nesbit et al. 1999). The MYC oncoprotein is a basic helix-loop-helix-leucine zipper (bHLHZIP) transcription factor that through dimerization with MAX protein binds to specific DNA elements ("E boxes") and modulates transcription of a wide variety of genes (for review, see Dang 1999;Grandori et al. 2000;Oster et al. 2002). The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli (Evan et al. 1992). MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence (Wang et al. 1998a;Greenberg et al. 1999;Wu et al. 1999). We reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target.Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene (Yu et al. 1996). The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair (Brosh and Bohr 2002;Saintigny et al. 2002). Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation (Shiratori et al. 1999). In order to delineate potential interactions between MYC and WRN in tumorigenesis, we determined whether WRN expression is modulated by MYC, and monitored cellular responses to MYC overexpression in the absence of WRN. The results indicated that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibr...

Evasion of apoptosis is critical in Myc-induced tumor progression. Here we report that cancer cells evade death under stress by activating calpain-mediated proteolysis of Myc. This generates Myc-nick, a cytoplasmic, transcriptionally inactive cleavage product of Myc. We found conversion of Myc into Myc-nick in cell lines and tissues derived from multiple cancers. In colon cancer, the production of Myc-nick is enhanced under stress conditions such as hypoxia and nutrient deprivation. Under these conditions, ectopic expression of Myc-nick promotes anchorage-independent growth and cell survival at least in part by promoting autophagy. Myc-nick also delays colon cancer cell death after treatment with chemotherapeutic drugs such as etoposide, cisplatin, and imatinib. Furthermore, colon cancer cells expressing a cleavage-resistant form of Myc undergo extensive apoptosis but are rescued by overexpression of Myc-nick. We also found that ectopic expression of Myc-nick results in the induction of the actin-bundling protein fascin, formation of filopodia, and increased cell motility-all mediators of tumor metastasis. Myc-nick-induced survival, autophagy, and motility require Myc box II (MBII), a region of Myc-nick that recruits acetyltransferases that in turn modify cytoplasmic proteins, including a-tubulin and ATG3. Our results suggest that Myc-nick-induced survival and motility contribute to colon cancer progression and metastasis.

In silico designed nucleic acid probes and primers often fail to achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. Here, we present a novel, on-the-fly method of tuning probe affinity and selectivity via the stoichiometry of auxiliary species, allowing independent and decoupled adjustment of hybridization yield for different probes in multiplexed assays. Using this method, we achieve near-continuous tuning of probe effective free energy (0.03 kcal·mol−1 granularity). As applications, we enforced uniform capture efficiency of 31 DNA molecules (GC content 0% – 100%), maximized signal difference for 11 pairs of single nucleotide variants, and performed tunable hybrid-capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples (FFPE).