Victor Sementchenko

RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Genome-wide analysis of estrogen receptor binding sites

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

Ets target genes: past, present and future.

Ets is a family of transcription factors present in species ranging from sponges to human. All family members contain an approximately 85 amino acid DNA binding domain, designated the Ets domain. Ets proteins bind to specific purine-rich DNA sequences with a core motif of GGAA/T, and transcriptionally regulate a number of viral and cellular genes. Thus, Ets proteins are an important family of transcription factors that control the expression of genes that are critical for several biological processes, including cellular proliferation, differentiation, development, transformation, and apoptosis. Here, we tabulate genes that are regulated by Ets factors and describe past, present and future strategies for the identification and validation of Ets target genes. Through definition of authentic target genes, we will begin to understand the mechanisms by which Ets factors control normal and abnormal cellular processes.

Biological function of unannotated transcription during the early development of Drosophila melanogaster

Many animal and plant genomes are transcribed much more extensively than current annotations predict. However, the biological function of these unannotated transcribed regions is largely unknown. Approximately 7% and 23% of the detected transcribed nucleotides during D. melanogaster embryogenesis map to unannotated intergenic and intronic regions, respectively. Based on computational analysis of coordinated transcription, we conservatively estimate that 29% of all unannotated transcribed sequences function as missed or alternative exons of well-characterized protein-coding genes. We estimate that 15.6% of intergenic transcribed regions function as missed or alternative transcription start sites (TSS) used by 11.4% of the expressed protein-coding genes. Identification of P element mutations within or near newly identified 5′ exons provides a strategy for mapping previously uncharacterized mutations to their respective genes. Collectively, these data indicate that at least 85% of the fly genome is transcribed and processed into mature transcripts representing at least 30% of the fly genome.

ETS2 function is required to maintain the transformed state of human prostate cancer cells.

The contribution of the ETS2 transcription factor to the transformed state in prostate cancer cells has been assessed. Northern blot analysis easily detects ETS2 in DU145 and PC3, high grade human prostate cell lines, but ETS2 is not present in lower grade LNCaP cells. Stable transfection of PC3 and DU145 prostate cell lines with an antisense ETS2 vector or with a dominant negative ETS2 mutant significantly reduced the ability of DU145 and PC3 cells to form large colonies in soft agar. Thus, the presence of ETS2 is positively correlated with a more transformed phenotype and blockage of ETS2 function can reduce transformed properties of prostate cancer cells.

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Dorsal–ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. Here, we attempt to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5′ exons were identified for at least 10 of the ≈100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units were identified, including two that contain known microRNAs, miR-1 and -9a. We discuss the potential functions of these recently identified genes and suggest that intronic enhancers are a common feature of the DV gene network.

SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion

The ETS1 transcription factor is a member of the Ets family of conserved sequence-specific DNA-binding proteins. ETS1 has been shown to play important roles in various cellular processes such as proliferation, differentiation, lymphoid development, motility, invasion and angiogenesis. These diverse roles of ETS1 are likely to be dependent on specific protein interactions. To identify proteins that interact with ETS1, a yeast two-hybrid screen was conducted. Here, we describe the functional interaction between SP100 and ETS1. SP100 protein interacts with ETS1 both in vitro and in vivo. SP100 is localized to nuclear bodies and ETS1 expression alters the nuclear body morphology in living cells. SP100 negatively modulates ETS1 transcriptional activation of the MMP1 and uPA promoters in a dose-dependent manner, decreases the expression of these endogenous genes, and reduces ETS1 DNA binding. Expression of SP100 inhibits the invasion of breast cancer cells and is induced by Interferon-α, which has been shown to inhibit the invasion of cancer cells. These data demonstrate that SP100 modulates ETS1-dependent biological processes.

Abstract 4826: Identification of low prevalence somatic mutations in heterogeneous tumor samples

High throughput sequencing enables the digital measurement of each allele in a DNA sample. This provides an ideal method to interrogate mutations present in heterogeneous samples such as solid tumors in which clonal selection or contamination with stroma can hinder the identification of important somatic mutations. We developed an ultra-deep targeted sequencing (UDT-Seq) assay to screen 46 cancer genes via microdroplet PCR (RainDance Technologies) and direct sequencing of the amplicons on the Illumina GA. This UDT-Seq library interrogates ∼86 kb of DNA located in cancer mutational hotspots (87% of all COSMIC mutations) and ∼29 kb located in exons sequenced in HapMap samples for the assay calibration and performance evaluation. We devised a statistical filtering of the mutations by using both experimental estimation and statistical modeling of the sequencing error rate. We measured the performance of our assay by processing 4 blends of 4 HapMap samples, interrogating 160 SNPs with known prevalence in each blend. The sensitivity and specificity of our method is >90% and >99% respectively for variants present at 5% prevalence or greater. We next interrogated 4 cancer samples (xenografts, 2 of which with matching primary samples) from 4 different tissues. We were able to detect low-prevalence somatic mutations in all samples of which some are well-known driver mutations. Interestingly, the mutational profile from primary to xenograft is conserved for one sample but different for another. Finally, we analyzed the robustness of the detection and prevalence measurement after performing whole-genome amplification on DNA extracted from fresh frozen and FFPE tissue using Single Primer Isothermal Amplification technology (SPIA® -NuGEN Technologies). Featuring a streamlined sample preparation to interrogate a large number of bases, this assay is well suited for clinical applications to study clonal selection in cancer progression or treatment with sub-optimal heterogeneous cancer samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4826. doi:10.1158/1538-7445.AM2011-4826