Integrating 5hmC and gene expression data to infer regulatory mechanisms.

Motivation: Epigenetic mechanisms are known to play a major role in breast cancer. However, the role of 5-hydroxymethylcytosine (5hmC) remains understudied. We hypothesize that 5hmC mediates redox regulation of gene expression in an aggressive subtype known as triple negative breast cancer (TNBC). To address this, our objective was to highlight genes that may be the target of this process by identifying redox-regulated, antioxidant-sensitive, gene-localized 5hmC changes associated with mRNA changes in TNBC cells. Results: We proceeded to develop an approach to integrate novel Pvu-sequencing and RNA-sequencing data. The result of our approach to merge genome-wide, high-throughput TNBC cell line datasets to identify significant, concordant 5hmC and mRNA changes in response to antioxidant treatment produced a gene set with relevance to cancer stem cell function. Moreover, we have established a method that will be useful for continued research of 5hmC in TNBC cells and tissue samples. Availability and implementation: Data are available at Gene Expression Omnibus (GEO) under accession number GSE103850. Contact: bollig@karmanos.org.

Cytogenomic profiling of breast cancer brain metastases reveals potential for repurposing targeted therapeutics.

Breast cancer brain metastases remain a significant clinical problem. Chemotherapy is ineffective and a lack of treatment options result in poor patient outcomes. Targeted therapeutics have proven to be highly effective in primary breast cancer, but lack of molecular genomic characterization of metastatic brain tumors is hindering the development of new treatment regimens. Here we contribute to fill this void by reporting on gene copy number variation (CNV) in 10 breast cancer metastatic brain tumors, assayed by array comparative genomic hybridization (aCGH). Results were compared to a list of cancer genes verified by others to influence cancer. Cancer gene aberrations were identified in all specimens and pathway-level analysis was applied to aggregate data, which identified stem cell pluripotency pathway enrichment and highlighted recurring, significant amplification of SOX2, PIK3CA, NTRK1, GNAS, CTNNB1, and FGFR1. For a subset of the metastatic brain tumor samples (n = 4) we compared patient-matched primary breast cancer specimens. The results of our CGH analysis and validation by alternative methods indicate that oncogenic signals driving growth of metastatic tumors exist in the original cancer. This report contributes support for more rapid development of new treatments of metastatic brain tumors, the use of genomic-based diagnostic tools and repurposed drug treatments.

(-)-Epicatechin combined with 8 weeks of treadmill exercise is associated with increased angiogenic and mitochondrial signaling in mice.

The purpose of this study was to conduct an 8 week endurance training program with and without (-)-epicatechin treatment and to determine whether there is a possible cumulative effect on protein markers of angiogenesis and mitochondrial biogenesis. Thirty-four 14-month old male mice (C57BL/6N) were randomized into four groups: control (C); (-)-epicatechin only ((-)-Epi); control with endurance training (CE); and (-)-epicatechin with endurance training ((-)-Epi-Ex). Mice in the training groups performed treadmill exercise for 8 weeks (5 × /week for 60 min/session), whereas mice in the (-)-epicatechin group received 1.0 mg/kg of body mass twice daily during the training period. At 8 weeks, distance ran on the treadmill increased by 46, 69, and 84% in the (-)-Epi, CE, and (-)-Epi-Ex groups, respectively compared to the control group (p < 0.001 for all comparisons). Furthermore, the (-)-Epi-Ex group had significantly higher exercise capacity than the (-)-Epi and CE group. For angiogenic regulators, the (-)-Epi-Ex group had significantly higher VEGF-R2 protein expression with a concomitant reduction in TSP-1 protein expression than the exercise group. Interestingly, FoxO1 protein expression was significantly reduced for all three experimental groups compared to the control group. Protein markers such as PGC-1ß and TFAM were significantly higher in the (-)-Epi-Ex group compared to the three other groups. These findings suggest that (-)-epicatechin treatment combined with 8 weeks of endurance training provide a cumulative effect on a number of angiogenic and mitochondrial signaling which functionally translates to enhanced exercise tolerance.

Gene expression profiling of replicative and induced senescence.

Cellular senescence is a cell cycle arrest accompanied by high expression of cyclin dependent kinase inhibitors which counteract overactive growth signals, which serves as a tumor suppressive mechanism. Senescence can be a result of telomere shortening (natural or replicative senescence) or DNA damage resulting from exogenous stressors (induced senescence). Here, we performed gene expression profiling through RNA-seq of replicative senescence, adriamycin-induced senescence, H2O2-induced senescence, and 5-aza-2-deoxycytidine-induced senescence in order to profile the pathways controlling various types of senescence. Overall, the pathways common to all 4 types of senescence were related to inflammation and the innate immune system. It was also evident that 5-aza-induced senescence mirrors natural replicative senescence due to telomere shortening. We also examined the prevalence of senescence-associated secretory phenotype (SASP) factors in the RNA-seq data, showing that it is a common characteristic of all 4 types of senescence. In addition, we could discriminate changes in gene expression due to quiescence during cellular senescence from those that were specific to senescence.

Modeling time-dependent transcription effects of HER2 oncogene and discovery of a role for E2F2 in breast cancer cell-matrix adhesion.

MOTIVATION: Oncogenes are known drivers of cancer phenotypes and targets of molecular therapies; however, the complex and diverse signaling mechanisms regulated by oncogenes and potential routes to targeted therapy resistance remain to be fully understood. To this end, we present an approach to infer regulatory mechanisms downstream of the HER2 driver oncogene in SUM-225 metastatic breast cancer cells from dynamic gene expression patterns using a succession of analytical techniques, including a novel MP grammars method to mathematically model putative regulatory interactions among sets of clustered genes. RESULTS: Our method highlighted regulatory interactions previously identified in the cell line and a novel finding that the HER2 oncogene, as opposed to the proto-oncogene, upregulates expression of the E2F2 transcription factor. By targeted gene knockdown we show the significance of this, demonstrating that cancer cell-matrix adhesion and outgrowth were markedly inhibited when E2F2 levels were reduced. Thus, validating in this context that upregulation of E2F2 represents a key intermediate event in a HER2 oncogene-directed gene expression-based signaling circuit. This work demonstrates how predictive modeling of longitudinal gene expression data combined with multiple systems-level analyses can be used to accurately predict downstream signaling pathways. Here, our integrated method was applied to reveal insights as to how the HER2 oncogene drives a specific cancer cell phenotype, but it is adaptable to investigate other oncogenes and model systems. AVAILABILITY AND IMPLEMENTATION: Accessibility of various tools is listed in methods; the Log-Gain Stoichiometric Stepwise algorithm is accessible at http://www.cbmc.it/software/Software.php.

Epigenetics of early-life lead exposure and effects on brain development.

The epigenetic machinery plays a pivotal role in the control of many of the body's key cellular functions. It modulates an array of pliable mechanisms that are readily and durably modified by intracellular or extracellular factors. In the fast-moving field of neuroepigenetics, it is emerging that faulty epigenetic gene regulation can have dramatic consequences on the developing CNS that can last a lifetime and perhaps even affect future generations. Mounting evidence suggests that environmental factors can impact the developing brain through these epigenetic mechanisms and this report reviews and examines the epigenetic effects of one of the most common neurotoxic pollutants of our environment, which is believed to have no safe level of exposure during human development: lead.

A survey of small RNAs in human sperm.

BACKGROUND: There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24,000 sncRNAs within each normal human spermatozoon. METHODS: RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18-30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS: Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈ 7%), Piwi-interacting piRNAs (≈ 17%), repeat-associated small RNAs (≈ 65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈ 11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS: A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization.

Genome-wide analysis of cancer/testis gene expression.

Cancer/Testis (CT) genes, normally expressed in germ line cells but also activated in a wide range of cancer types, often encode antigens that are immunogenic in cancer patients, and present potential for use as biomarkers and targets for immunotherapy. Using multiple in silico gene expression analysis technologies, including twice the number of expressed sequence tags used in previous studies, we have performed a comprehensive genome-wide survey of expression for a set of 153 previously described CT genes in normal and cancer expression libraries. We find that although they are generally highly expressed in testis, these genes exhibit heterogeneous gene expression profiles, allowing their classification into testis-restricted (39), testis/brain-restricted (14), and a testis-selective (85) group of genes that show additional expression in somatic tissues. The chromosomal distribution of these genes confirmed the previously observed dominance of X chromosome location, with CT-X genes being significantly more testis-restricted than non-X CT. Applying this core classification in a genome-wide survey we identified >30 CT candidate genes; 3 of them, PEPP-2, OTOA, and AKAP4, were confirmed as testis-restricted or testis-selective using RT-PCR, with variable expression frequencies observed in a panel of cancer cell lines. Our classification provides an objective ranking for potential CT genes, which is useful in guiding further identification and characterization of these potentially important diagnostic and therapeutic targets.

Simplified ontologies allowing comparison of developmental mammalian gene expression.

Model organisms represent an important resource for understanding the fundamental aspects of mammalian biology. Mapping of biological phenomena between model organisms is complex and if it is to be meaningful, a simplified representation can be a powerful means for comparison. The Developmental eVOC ontologies presented here are simplified orthogonal ontologies describing the temporal and spatial distribution of developmental human and mouse anatomy. We demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse.

Mice and men: their promoter properties.

Using the two largest collections of Mus musculus and Homo sapiens transcription start sites (TSSs) determined based on CAGE tags, ditags, full-length cDNAs, and other transcript data, we describe the compositional landscape surrounding TSSs with the aim of gaining better insight into the properties of mammalian promoters. We classified TSSs into four types based on compositional properties of regions immediately surrounding them. These properties highlighted distinctive features in the extended core promoters that helped us delineate boundaries of the transcription initiation domain space for both species. The TSS types were analyzed for associations with initiating dinucleotides, CpG islands, TATA boxes, and an extensive collection of statistically significant cis-elements in mouse and human. We found that different TSS types show preferences for different sets of initiating dinucleotides and cis-elements. Through Gene Ontology and eVOC categories and tissue expression libraries we linked TSS characteristics to expression. Moreover, we show a link of TSS characteristics to very specific genomic organization in an example of immune-response-related genes (GO:0006955). Our results shed light on the global properties of the two transcriptomes not revealed before and therefore provide the framework for better understanding of the transcriptional mechanisms in the two species, as well as a framework for development of new and more efficient promoter- and gene-finding tools.