Just over 25 years ago, MYC, the human homologue of a retroviral oncogene, was identified. Since that time, MYC research has been intense and the advances impressive. On reflection, it is astonishing how each incremental insight into MYC regulation and function has also had an impact on numerous biological disciplines, including our understanding of molecular oncogenesis in general. Here we chronicle the major advances in our understanding of MYC biology, and peer into the future of MYC research.

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The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of ␦-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6 -8 maintain an ␣-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.Programmed cell death or apoptosis is a normal physiological form of cell death essential for successful embryonic development and the maintenance of cellular homeostasis (1-3). The BCL-2 family is comprised of pro-as well as anti-apoptotic proteins and constitutes critical points in the apoptosis pathway. BID is a pro-apoptotic member of the "BH3-only" subset of the BCL-2 family proteins (4) that interconnects extrinsic pathway TNFR1 and Fas death signals to the mitochondrial amplification of the intrinsic pathway. Engagement of TNFR1 and Fas activates caspase-8 that cleaves p22 BID within an unstructured loop generating an N-terminal 7-kDa fragment and a C-terminal 15-kDa fragment (5-7). Cleaved BID then undergoes post-translational modification by myristoylation at a newly generated N-terminal glycine of the p15-kDa fragment (p15 BID or tBID) (8). Myristoylation of the BID complex p7/ myr-p15 serves as a molecular switch facilitating the targeting of BID to the mitochondrion. tBID localizes to mitochondrial contact sites where cardiolipin has been reported to exist (9 -11). Targeted p15 BID triggers the homo-oligomerization of multidomain pro-apoptotic BAX or BAK in the mitochondrial outer membrane (MOM) 1 (12-15). This results in the permeabilization of the MOM and release of intermembrane space proteins, including cytochrome c which forms an apoptosome complex with Apaf-1 and caspase-9 to activate effector caspases (2,3,16,17). In addition to the role of tBID in activation of the critical gateway proteins BAX and BAK, targeted p15 BID also triggers remodeling of the inner membrane and the cristae structure of the mitochondria in a BAX-, BAK-independent manner, resulting in the mobilization of the majority of cytochrome c within the cristae that is stored in the intermembrane space of the organelle (18). The actions of tBID are antagonized by anti-apoptotic proteins BCL-2 or BCL-X L (5,7,19). Recently, it has been reported that BID and p15 BID are capable of binding distinct lipids, which led to the hypothesis that BID might play a role in the transport and re...

Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack. Cancer Res; 73(21); 6504-15. Ó2013 AACR.

Background Temperate phages influence the density, diversity and function of bacterial populations. Historically, they have been described as carriers of toxins. More recently, they have also been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. Despite recent advances in studying prophages using non-targeted sequencing approaches, methodological challenges in identifying inducible prophages in bacterial genomes and quantifying their activity have limited our understanding of prophage-host interactions. Results We present methods for using high-throughput sequencing data to locate inducible prophages, including those previously undiscovered, to quantify prophage activity and to investigate their replication. We first used the well-established Salmonella enterica serovar Typhimurium/p22 system to validate our methods for (i) quantifying phage-to-host ratios and (ii) accurately locating inducible prophages in the reference genome based on phage-to-host ratio differences and read alignment alterations between induced and non-induced prophages. Investigating prophages in bacterial strains from a murine gut model microbiota known as Oligo-MM12 or sDMDMm2, we located five novel inducible prophages in three strains, quantified their activity and showed signatures of lateral transduction potential for two of them. Furthermore, we show that the methods were also applicable to metagenomes of induced faecal samples from Oligo-MM12 mice, including for strains with a relative abundance below 1%, illustrating its potential for the discovery of inducible prophages also in more complex metagenomes. Finally, we show that predictions of prophage locations in reference genomes of the strains we studied were variable and inconsistent for four bioinformatic tools we tested, which highlights the importance of their experimental validation. Conclusions This study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity. The ability to generate such quantitative information will be critical in helping us to gain better insights into the factors that determine phage activity and how prophage-bacteria interactions influence our microbiome and impact human health.

<p>PDF file, 498K, Supplementary Figures S1-6 Supplementary Figure S1: Phosphorylation mutants increase Myc-induced transformation in MCF10A cells. Supplementary Figure S2: Phosphorylation mutants increase Myc-induced transformation in SH-EP cells. Supplementary Figure S3: Phosphorylation mutants do not effect MYC protein stability. Supplementary Figure S4: Serum starvation of MCF10A-GFP and MCF10A-MYC cells. Supplementary Figure S5: MYC protein expression through morphogenesis of MCF10A cells in 3D culture. Supplementary Figure S6: Characterization of day 4 acini for mRNA expression array analysis.</p>

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<p>XLS file, 403K, Expression comparison between wild-type MYC and MYC phosphorylation mutants (from Venn diagram)</p>