Differential Analysis of Ovarian and Endometrial Cancers Identifies a Methylator Phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease

Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus

Silencers and enhancer-blockers (EBs) are cis-acting, negative regulatory elements (NREs) that control interactions between promoters and enhancers. Although relatively uncharacterized in terms of biological mechanisms, these elements are likely to be abundant in the genome. We developed an experimental strategy to identify silencers and EBs using transient transfection assays. A known insulator and EB from the chicken beta-globin locus, cHS4, served as a control element for these assays. We examined 47 sequences from a 1.8-Mb region of human chromosome 7 for silencer and EB activities. The majority of functional elements displayed directional and promoter-specific activities. A limited number of sequences acted in a dual manner, as both silencers and EBs. We examined genomic data, epigenetic modifications, and sequence motifs within these regions. Strong silencer elements contained a novel CT-rich motif, often in multiple copies. Deletion of the motif from three regions caused a measurable loss of silencing ability in these sequences. Moreover, five duplicate occurrences of this motif were identified in the cHS4 insulator. These motifs provided an explanation for an uncharacterized silencing activity we measured in the insulator element. Overall, we identified 15 novel NREs, which contribute new insights into the prevalence and composition of sequences that negatively regulate gene expression

Bidirectional Promoters as Important Drivers for the Emergence of Species-Specific Transcripts

<div><p>The diversification of gene functions has been largely attributed to the process of gene duplication. Novel examples of genes originating from previously untranscribed regions have been recently described without regard to a unifying functional mechanism for their emergence. Here we propose a model mechanism that could generate a large number of lineage-specific novel transcripts in vertebrates through the activation of bidirectional transcription from unidirectional promoters. We examined this model <i>in silico</i> using human transcriptomic and genomic data and identified evidence consistent with the emergence of more than 1,000 primate-specific transcripts. These are transcripts with low coding potential and virtually no functional annotation. They initiate at less than 1 kb upstream of an oppositely transcribed conserved protein coding gene, in agreement with the generally accepted definition of bidirectional promoters. We found that the genomic regions upstream of ancestral promoters, where the novel transcripts in our dataset reside, are characterized by preferential accumulation of transposable elements. This enhances the sequence diversity of regions located upstream of ancestral promoters, further highlighting their evolutionary importance for the emergence of transcriptional novelties. By applying a newly developed test for positive selection to transposable element-derived fragments in our set of novel transcripts, we found evidence of adaptive evolution in the human lineage in nearly 3% of the novel transcripts in our dataset. These findings indicate that at least some novel transcripts could become functionally relevant, and thus highlight the evolutionary importance of promoters, through their capacity for bidirectional transcription, for the emergence of novel genes.</p> </div

Assessing positive selection in the third exon of the AK094354.

<p>(<b>A</b>) Alignment of the 70-bp human exon to the AluJb consensus sequence identifies positions 35–115 in the AluJb consensus as the source of the exon. (<b>B</b>) Alignment of the AluJb-derived exon between human, chimp and macaque orthologs. Positions of human specific changes are shown in red, and vertical bars separate the adjacent flanking consensus splicing dinucleotides. (<b>C</b>) Distribution of human-specific changes in 70,275 AluJb intergenic homologs that correspond to the 35–115 AluJb consensus region. (<b>D</b>) To take local mutation bias into account, the values are expressed as excess over local human-specific substitution rates (right panel; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057323#s4" target="_blank">Methods</a>). Red arrows correspond to values observed for the 70-bp exon of interest, and represent significantly high substitution rates (<i>P</i> = 0.006 and <i>P</i> = 0.004, respectively) in both cases.</p

Low conservation of PINTs illustrated by phastCons scores.

<p>(<b>A</b>) Distribution of transcript-wide phastCons average-scores for PINTs (red, median 0.026), cncRNAs (black, median 0.06), and anchors (blue, median 0.535). (<b>B</b>) Distribution of phastCons scores for all splice sites for PINTs, cncRNAs, and anchors (medians of 5×10−4, 0.0025, and 0.9995, respectively).</p

Experimental evidence for the splicing of the AluJb-derived exon 3 in AK094354.

<p>(<b>A</b>) The splicing efficiency of the AluJb-derived exon was tested in a minigene splicing assay both with the human sequence, as well as with the chimp and macaque orthologs. The upper band (which includes the 70-bp exon) is the strongest in human both visually and quantitatively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057323#pone.0057323.s015" target="_blank">Fig. S15</a>), whereas the lower band corresponds to the exon being skipped. The Invitrogen 100-bp DNA ladder (L) was used as a marker. (<b>B</b>) RT-PCR of the transcript in 12 human tissues: 1 - brain, 2 - spleen, 3 - muscle, 4 - placenta, 5 - heart, 6 - liver, 7 - lung, 8 - stomach, 9 - kidney, 10 - intestine, 11 - testis, 12 - colon. Two dominant bands (581 and 466 bps) were found in ten tissues (no product observed in placenta or heart), corresponding to isoforms with alternatively spliced exon 2 of the transcript (115 bps). A shorter band can be observed for testes, which corresponds to non-specific amplification from chromosome 6 (verified by sequencing).</p

Assesing the strength and TE origin of SSs.

<p>(<b>A</b>) The acceptor (3′) SSs used by PINTs exhibit higher SplicePort scores (red; median 0.37) than background splice signals (black; median −3.15, <i>P</i> = 0, MWU test), but lower than score of SSs used by cncRNAs (green; median 0.41, <i>P</i> = 0.07) and anchors (blue; median 0.96, <i>P</i> = 4.5×10⁻¹⁴⁸). Dashed vertical lines indicate median values of corresponding distributions. (<b>B</b>) Random sets of 1,939 “AG” dinucleotides (10,000 replicates) were selected to match the score distribution of acceptor SSs used by PINTs to determine the expected fraction of TE-derived SSs. The confirmation of the score distribution fit is provided by the non-significant difference between the median of PINT acceptor SS scores (0.37) and random samples (<i>P</i> = 0.19). (<b>C</b>) Using the same sets as in (B), we show that the fraction of TE-derived acceptor SSs in PINTs (37.9%, red arrow) is not significantly different (<i>P</i> = 0.16) from expectation.</p

SplicePort scores for SSs flanking the third exon of the AK094354 transcript.

<p>Chimp and macaque scores were computed for sequences orthologous to human SS (alignments of the exonic regions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057323#pone-0057323-g005" target="_blank">Fig. 5B</a>). The highest values were observed in human, and the lowest in macaque. Score percentiles were computed based on the original set of SSs used for SplicePort training.</p