John D. Watson

Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression.

The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Mycs importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.

Spatial genomic heterogeneity within localized, multifocal prostate cancer

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Identifying Genes Regulated in a Myc-dependent Manner

The c-myc proto-oncogene can direct a diverse array of biological activities, including cell cycle progression, apoptosis, and differentiation. It is believed that Myc can affect this wide variety of activities by functioning as a regulator of gene transcription, although few targets have been identified to date. To delineate the molecular program regulated downstream of Myc, we used a cDNA microarray approach and identified 52 putative targets out of >6000 cDNAs analyzed. To further distinguish the subset of genes whose regulation was dependent upon Myc per se from those regulated in response to activation of general mitogenic or apoptotic programs, the putative cDNA targets were then screened by a series of assays. By this approach 37 putative targets were ruled out and 15 Myc target genes were uncovered. Interestingly, comparing our results with other high throughput screens reveals that certain putative Myc targets previously reported are shown not to be regulated downstream of Myc (e.g. ribosomal proteins, HSP90β), whereas others are further supported by our analyses (e.g. pdgfβr, nucleolin). The identity of genes specifically regulated downstream of Myc provides the critical tools required to understand the role Myc holds in the transformation process and to delineate how Myc functions as a regulator of gene transcription.

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome

An effective tool for the global analysis of both DNA methylation status and protein–chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20 736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein–chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity

The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD₅₀ of > 9600 μg/kg for H/W rats is higher than for any other wild-type mammal known. We previously showed that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effects of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 μg/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome.

Systematic evaluation of medium-throughput mRNA abundance platforms

Profiling of mRNA abundances with high-throughput platforms such as microarrays and RNA-seq has become an important tool in both basic and biomedical research. However, these platforms remain prone to systematic errors and have challenges in clinical and industrial applications. As a result, it is standard practice to validate a subset of key results using alternate technologies. Similarly, clinical and industrial applications typically involve transitions from a high-throughput discovery platform to medium-throughput validation ones. These medium-throughput validation platforms have high technical reproducibility and reduced sample input needs, and low sensitivity to sample quality (e.g., for processing FFPE specimens). Unfortunately, while medium-throughput platforms have proliferated, there are no comprehensive comparisons of them. Here we fill that gap by comparing two key medium-throughput platforms--NanoStrings nCounter Analysis System and ABIs OpenArray System--to gold-standard quantitative real-time RT-PCR. We quantified 38 genes and positive and negative controls in 165 samples. Signal:noise ratios, correlations, dynamic range, and detection accuracy were compared across platforms. All three measurement technologies showed good concordance, but with divergent price/time/sensitivity trade-offs. This study provides the first detailed comparison of medium-throughput RNA quantification platforms and provides a template and a standard data set for the evaluation of additional technologies.

ShatterProof: operational detection and quantification of chromothripsis

BackgroundChromothripsis, a newly discovered type of complex genomic rearrangement, has been implicated in the evolution of several types of cancers. To date, it has been described in bone cancer, SHH-medulloblastoma and acute myeloid leukemia, amongst others, however there are still no formal or automated methods for detecting or annotating it in high throughput sequencing data. As such, findings of chromothripsis are difficult to compare and many cases likely escape detection altogether.ResultsWe introduce ShatterProof, a software tool for detecting and quantifying chromothriptic events. ShatterProof takes structural variation calls (translocations, copy-number variations, short insertions and loss of heterozygosity) produced by any algorithm and using an operational definition of chromothripsis performs robust statistical tests to accurately predict the presence and location of chromothriptic events. Validation of our tool was conducted using clinical data sets including matched normal, prostate cancer samples in addition to the colorectal cancer and SCLC data sets used in the original description of chromothripsis.ConclusionsShatterProof is computationally efficient, having low memory requirements and near linear computation time. This allows it to become a standard component of sequencing analysis pipelines, enabling researchers to routinely and accurately assess samples for chromothripsis. Source code and documentation can be found at http://search.cpan.org/~sgovind/Shatterproof.

TCDD dysregulation of 13 AHR-target genes in rat liver

Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long-Evans (Turku/AB; L-E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000μg/kg at 19h after TCDD exposure and time points ranging from 1.5 to 384h after exposure to 100μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose-response analysis, none had an ED50 equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10-100 fold higher, in at least one strain (Ahrr (L-E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L-E), Inmt (both), Nfe2l2 (L-E), Nqo1 (L-E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities.

Inter-strain heterogeneity in rat hepatic transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been the subject of intense study for decades. It is now clear that essentially all TCDD-induced toxicities are mediated by DNA-protein interactions involving the Aryl Hydrocarbon Receptor (AHR). Nevertheless, it remains unknown which AHR target genes cause TCDD toxicities. Several groups, including our own, have developed rodent model systems to probe these questions. mRNA expression profiling of these model systems has revealed significant inter-species heterogeneity in rodent hepatic responses to TCDD. It has remained unclear if this variability also exists within a species, amongst rodent strains. To resolve this question, we profiled the hepatic transcriptomic response to TCDD of diverse rat strains (L-E, H/W, F344 and Wistar rats) and two lines derived from L-E×H/W crosses, at consistent age, sex, and dosing (100 μg/kg TCDD for 19 h). Using this uniquely consistent dataset, we show that the majority of TCDD-induced alterations in mRNA abundance are strain/line-specific: only 11 genes were affected by TCDD across all strains, including well-known dioxin-responsive genes such as Cyp1a1 and Nqo1. Our analysis identified two novel universally dioxin-responsive genes as well as 4 genes induced by TCDD in dioxin-sensitive rats only. These 6 genes are strong candidates to explain TCDD-related toxicities, so we validated them using 152 animals in time-course (0 to 384 h) and dose-response (0 to 3000 μg/kg) experiments. This study reveals that different rat strains exhibit dramatic transcriptional heterogeneity in their hepatic responses to TCDD and that inter-strain comparisons can help identify candidate toxicity-related genes.

Identification of a novel Brain Derived Neurotrophic Factor (BDNF)-inhibitory factor: Regulation of BDNF by Teneurin C-terminal Associated Peptide (TCAP)-1 in immortalized embryonic mouse hypothalamic cells

The teneurins are a family of four large transmembrane proteins that are highly expressed in the central nervous system (CNS) where they have been implicated in development and CNS function. At the tip of the carboxyl terminus of each teneurin lies a 43-amino acid sequence, that when processed, could liberate an amidated 41-residue peptide. We have called this region the teneurin C-terminal associated peptide (TCAP). Picomolar concentrations of the synthetic version of TCAP-1 inhibit stress-induced cocaine reinstatement in rats. Because cocaine-seeking is associated with increased brain derived neurotrophic factor (BDNF) in the brain, we examined whether synthetic mouse TCAP-1 has the potential to regulate BDNF expression in immortalized mouse neurons. Immortalized mouse neurons (N38; mHypoE38) show strong FITC-labeled [K(8)]-TCAP-1 uptake and BDNF labeling in the cytosol. Moreover, FITC-labeled [K(8)]-TCAP-1 bound competitively to membrane fractions. In culture, the labeled TCAP-1 peptide could be detected on cell membranes within 15 min and subsequently became internalized in the cytosol and trafficked toward the nucleus. Administration of 10(-8)M unlabeled TCAP-1 to cultures of the N38 cells resulted in a significant decrease of total cell BDNF immunoreactivity over 4h as determined by western blot and ELISA analyses. Real-time PCR, utilizing primers to the various BDNF transcripts showed a significant decline of promoter IIB- and VI-driven transcripts. Taken together, these studies indicated that in vitro, TCAP-1 induces a significant decline in BDNF transcription and protein labeling in embyronic mouse immortalized hypothalamic neurons. Thus, TCAP-1 may act as a novel BDNF inhibitory factor.