Mammalian c-Myc is a member of a small family of three related proto-oncogenic transcription factors. c-Myc has an unusually broad array of regulatory functions, which include roles in cell cycle and apoptosis, a variety of metabolic functions, cell differentiation, senescence and stem cell maintenance. c-Myc modulates the expression of a very large number of genes, but the magnitude of the majority of the regulatory effects is only two-fold or less. c-Myc can both activate and repress the promoters of its target genes. Identification of genes directly regulated by c-Myc has been an enduring question in the field. We report here microarray expression profiling of a high resolution time course of c-Myc induction, using fibroblast cells in which c-Myc activity can be modulated from null to physiological. The c-Myc transcriptome data set presented is the largest reported to date with 4,186 differentially regulated genes (1,826 upregulated, 2,360 downregulated, 1% FDR). The gene expression patterns fit well with the known biological functions of c-Myc. We describe several novel findings and present tools for further data mining. Although the mechanisms of transcriptional activation by c-Myc are well understood, how c-Myc represses an even greater number of genes remains incompletely described. One mechanism involves the binding of c-Myc to other, positively acting transcription factors and interfering with their activities. We identified rapid-response genes likely to be direct c-Myc targets and analyzed the promoters of the repressed genes to identify transcription factors that could be targets of c-Myc repression.

Summary

Chromatin structure affects the accessibility of DNA to transcription, repair and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole genome tiling microarrays of activation and repressive chromatin marks, whole genome transcriptional microarrays and single cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome and islands of facultative heterochromatin dispersed throughout the genome. With age there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single cell immunohistochemistry that show changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair and DNA replication. The combination of genome-wide approaches such as whole genome chromatin immunoprecipitation and transcriptional studies in conjunction with single cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

Environmental and genetic interventions extend health span in a range of organisms by triggering changes in different specific but complementary pathways. We investigated the gene expression changes that occur across species when health span is extended via different interventions. To perform this comparison using heterogeneous datasets from different measurement platforms and organisms, we developed a novel non-parametric methodology that can detect statistical significance of overlaps in ranked lists of genes, and estimate the number of genes with a common expression profile. By comparing genetic and environmental interventions that consistently lead to increased health span in invertebrates and vertebrates we built a conserved health span signature and described how such a signature depends on tissue type. Furthermore, we examined the relationship between calorie restriction and resveratrol administration and for the first time, identified common gene and pathway changes in calorie restriction and resveratrol in both invertebrates and mammals. Our approach can thus be used to explore and better define the relationships between highly complex biological phenomena, in this case those that affect the health and longevity.

A multiple comparison approach using whole genome transcriptional arrays was used to identify genes and pathways involved in calorie restriction/dietary restriction (DR) life span extension in Drosophila. Starting with a gene centric analysis comparing the changes in common between DR and two DR related molecular genetic life span extending manipulations, Sir2 and p53, lead to a molecular confirmation of Sir2 and p53's similarity with DR and the identification of a small set of commonly regulated genes. One of the identified upregulated genes, takeout, known to be involved in feeding and starvation behavior, and to have sequence homology with Juvenile Hormone (JH) binding protein, was shown to directly extend life span when specifically overexpressed. Here we show that a pathway centric approach can be used to identify shared physiological pathways between DR and Sir2, p53 and resveratrol life span extending interventions. The set of physiological pathways in common among these life span extending interventions provides an initial step toward defining molecular genetic and physiological changes important in life span extension. The large overlap in shared pathways between DR, Sir2, p53 and resveratrol provide strong molecular evidence supporting the genetic studies linking these specific life span extending interventions.

A major challenge in translating the positive effects of dietary restriction (DR) for the improvement of human health is the development of therapeutic mimics. One approach to finding DR mimics is based upon identification of the proximal effectors of DR life span extension. Whole genome profiling of DR in Drosophila shows a large number of changes in gene expression, making it difficult to establish which changes are involved in life span determination as opposed to other unrelated physiological changes. We used comparative whole genome expression profiling to discover genes whose change in expression is shared between DR and two molecular genetic life span extending interventions related to DR, increased dSir2 and decreased Dmp53 activity. We find twenty-one genes shared among the three related life span extending interventions. One of these genes, takeout, thought to be involved in circadian rhythms, feeding behavior and juvenile hormone binding is also increased in four other life span extending conditions: Rpd3, Indy, chico and methuselah. We demonstrate takeout is involved in longevity determination by specifically increasing adult takeout expression and extending life span. These studies demonstrate the power of comparative whole genome transcriptional profiling for identifying specific downstream elements of the DR life span extending pathway.

Background

We investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation.

Methodology/Principal Findings

The hepatic cells were exposed to rapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoter regions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases.

Conclusions/Significance

There is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.

The c-myc proto-oncogene is rapidly activated by serum and regulates genes involved in metabolism and cell cycle progression. This gene is thereby uniquely poised to coordinate both the metabolic and cell cycle regulatory events required for cell cycle entry. However, this function of Myc has not been evaluated. Using a rat fibroblast model of isogenic cell lines, myc−/−, myc+/−, myc+/+ and myc−/− cells with an inducible c-myc transgene (mycER), we show that the Myc protein programs cells to utilize both oxidative phosphorylation and glycolysis to drive cell cycle progression. We demonstrate this coordinate regulation of metabolic networks is essential, as specific inhibitors of these pathways block Myc-induced proliferation. Metabolic events temporally correlated with cell cycle entry include increased oxygen consumption, mitochondrial function, pyruvate and lactate production, and ATP generation. Treatment of normal cells with inhibitors of oxidative phosphorylation recapitulates the myc −/−phenotype, resulting in impaired cell cycle entry and reduced metabolism. Combined with a kinetic expression profiling analysis of genes linked to mitochondrial function, our study indicates that Myc's ability to coordinately regulate the mitochondrial metabolic network transcriptome is required for rapid cell cycle entry. This function of Myc may underlie the pervasive presence of Myc in many human cancers.

Background

The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.

Methodology/Principal Findings

Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.

Conclusions/Significance

Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

Background

Significance analysis at single gene level may suffer from the limited number of samples and experimental noise that can severely limit the power of the chosen statistical test. This problem is typically approached by applying post hoc corrections to control the false discovery rate, without taking into account prior biological knowledge. Pathway or gene ontology analysis can provide an alternative way to relax the significance threshold applied to single genes and may lead to a better biological interpretation.

Results

Here we propose a new analysis method based on the study of networks of pathways. These networks are reconstructed considering both the significance of single pathways (network nodes) and the intersection between them (links).

We apply this method for the reconstruction of networks of pathways to two gene expression datasets: the first one obtained from a c-Myc rat fibroblast cell line expressing a conditional Myc-estrogen receptor oncoprotein; the second one obtained from the comparison of Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia derived from bone marrow samples.

Conclusion

Our method extends statistical models that have been recently adopted for the significance analysis of functional groups of genes to infer links between these groups. We show that groups of genes at the interface between different pathways can be considered as relevant even if the pathways they belong to are not significant by themselves.

Exon centric microarrays were used to resolve the calcium-modulated gene expression response into transcript-level an exon-level regulation.

Background:

Neuronal cells respond to changes in intracellular calcium ([Ca2+]i) by affecting both the abundance and architecture of specific mRNAs. Although calcium-induced transcription and transcript variation have both been recognized as important sources of gene regulation, the interplay between these two phenomena has not been evaluated on a genome-wide scale.

Results:

Here, we show that exon-centric microarrays can be used to resolve the [Ca2+]i-modulated gene expression response into transcript-level and exon-level regulation. Global assessments of affected transcripts reveal modulation within distinct functional gene categories. We find that transcripts containing calcium-modulated exons exhibit enrichment for calcium ion binding, calmodulin binding, plasma membrane associated, and metabolic proteins. Additionally, we uncover instances of regulated exon use in potassium channels, neuroendocrine secretory proteins and metabolic enzymes, and demonstrate that regulated changes in exon expression give rise to distinct transcript variants.

Conclusion:

Our findings connect extracellular stimuli to specific exon behavior, and suggest that changes in transcript and exon abundance are reflective of a coordinated gene expression response to elevated [Ca2+]i. The technology we describe here lends itself readily to the resolution of stimulus-induced gene expression at both the transcript and exon levels.

Time course gene expression experiments are a popular means to infer co-expression. Many methods have been proposed to cluster genes or to build networks based on similarity measures of their expression dynamics. In this paper we apply a correlation based approach to network reconstruction to three datasets of time series gene expression following system perturbation: 1) Conditional, Tamoxifen dependent, activation of the cMyc proto-oncogene in rat fibroblast; 2) Genomic response to nutrition changes in D. melanogaster; 3) Patterns of gene activity as a consequence of ageing occurring over a life-span time series (25y–90y) sampled from T-cells of human donors.

We show that the three datasets undergo similar transitions from an "uncorrelated" regime to a positively or negatively correlated one that is symptomatic of a shift from a "ground" or "basal" state to a "polarized" state.

In addition, we show that a similar transition is conserved at the pathway level, and that this information can be used for the construction of "meta-networks" where it is possible to assess new relations among functionally distant sets of molecular functions.