TU Pairs Searched For

<p>We defined a <i>cis–</i>antisense pair as two oppositely transcribed TUs that share at least 20 bp of exon sequence, a non-exon-overlapping antisense pair as two oppositely transcribed TUs that overlap by at least 20 bp, but not within exons, and a bidirectionally promoted pair as two divergently transcribed TUs that overlap by less than 20 bp and are less than 1,000 bp apart.</p

Landmark Sequence Composition of Bidirectional Promoters

<p>We defined the midpoint of a bidirectional promoter as the midpoint between the most 5′ TSS in each of the two divergently oriented TCs defining the bidirectional promoter. Sequences corresponding to the region spanned by the TCs were extracted from the genomic plus strand. All bidirectional promoter sequences were aligned at their midpoint and the logo created with WebLogo [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020047#pgen-0020047-b049" target="_blank">49</a>]. The logo displays the four nucleotides ranked by their frequency at each position, so that more common nucleotides appear above less common ones. The charts above the logo show the distribution of CAGE tag 5′-ends mapping to the plus strand (upper chart) and minus strand (lower chart) around bidirectional promoter midpoints. The CAGE tag distribution was computed as the sum of tag counts at each position over all bidirectional promoters. The peak of nearly 5,000 tags on the plus strand is due to the <i>Rps2</i> gene, which appears to be most highly expressed from a single TSS.</p

A Five-TU Chain on Mouse Chromosome 15

<p>TUs on the genomic plus and minus strands are shown in dark gray and light gray, respectively (boxes represent exons). CpG islands are shown as black boxes. From left to right, the chain contains a member of the aminoacyl tRNA transferase class II family <i>(D330001F17Rik),</i> which has two <i>cis–</i>antisense transcripts: fully overlapping (cDNA AK034666) and convergent <i>(Bop1)</i>. The latter encodes a ribosome biogenesis protein and shares a CpG-island bidirectional promoter with the heat-shock-induced transcription factor 1 gene <i>(Hsf1)</i>. <i>Hsf1,</i> in turn, is convergently <i>cis–</i>antisense to the diacylglycerol O-acyltransferase 1 gene <i>(Dgat1)</i>.</p

Estimating the Extent and Conservation of Antisense Transcription

<div><p>(A and B) Estimation of proportion of TUs involved in <i>cis–</i>antisense pairs. Open circles indicate the fraction of all human TUs on the plus strand (A) and all mouse TUs on the plus strand (B) that were found to be involved in <i>cis–</i>antisense pairs when the minus-strand TUs were recomputed starting from random transcript sequence samples of different sizes. Filled circles represent the full datasets based on all available transcript sequences. The saturation curves (see Equation 1) indicated by the lines fit almost perfectly to the sampled data. Fitted human and mouse saturation curves approach 0.45 and 0.43, respectively, as the number of transcript sequences increases, indicating that more than 40% of all TUs might be involved in <i>cis–</i>antisense pairs. Similar estimates were obtained by other sampling approaches (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020047#pgen-0020047-sg003" target="_blank">Figure S3</a>).</p> <p>(C) Estimation of the proportion of human <i>cis–</i>antisense pairs that are conserved in mouse. Open circles indicate the proportion of human <i>cis–</i>antisense pairs found to be conserved in mouse when the full human dataset was compared to mouse datasets recomputed from random mouse transcript sequence samples of different sizes. The same type of saturation curve as in (A) was fitted to the data. Here, a model with <i>c</i> = 1 (i.e., hyperbolic saturation) was preferable as it provided an equally good fit while being simpler. The fitted curve approaches 0.25 as the number of mappings grows, indicating that about 25% of human <i>cis–</i>antisense pairs are conserved in mouse.</p></div

Members of <i>cis–</i>Antisense Pairs Have Positively Correlated Expression Profiles More Often than Expected by Chance

<p>Out of 242 murine <i>cis–</i>antisense pairs with expression data for 61 tissues, 17 showed significant positive correlation across the entire set of tissues after correction for multiple testing, and no pairs showed significant negative correlation (red squares). The same test was applied to 10,000 sets of 242 random TU pairs (box plots, with circles indicating outliers), demonstrating that members of <i>cis–</i>antisense pairs have positively correlated expression profiles more often than expected by chance.</p

TSS Variability at the <i>Ddx49</i>/<i>Cope</i> Bidirectional Promoter in Mouse

<div><p>(A) The charts show the distribution of CAGE tag 5′-ends over the first five exons of each of the two genes <i>Ddx49</i> and <i>Cope,</i> and over their intergenic region. CAGE tag mappings indicate that transcription of <i>Cope</i> can start within two wide regions in the first exon of the gene. The initial part of this first exon (hatched) has support from several ESTs, but no cDNA sequences. The three large TCs at the <i>Ddx49</i>/<i>Cope</i> locus span 79, 114, and 150 bp, indicating great variability of transcriptional initiation within each cluster. To confirm the existence of such variability by qRT-PCR, primers (connected boxes) were designed to measure expression of selected regions of the <i>Ddx49</i> (primer pairs A1–A4) and <i>Cope</i> (primer pairs B1–B5) transcripts.</p> <p>(B) Detailed view of CAGE tag frequencies and primer locations over the three transcription initiation regions indicated by CAGE tags. Gray lines show cumulative CAGE tag frequencies.</p> <p>(C) Expression levels of different regions of the <i>Ddx49</i> and <i>Cope</i> transcripts in adult brain RNA as measured by qRT-PCR. Primer pairs A1 and A2 confirmed low level of expression of the longest <i>Ddx49</i> transcripts indicated by CAGE (copy numbers in 12.5 ng of total RNA were 3.2 [standard deviation = 1.1] and 5.1 [standard deviation = 3.0] for A1 and A2, respectively). Primer pair B1 confirmed transcription of <i>Cope</i> from upstream of the canonical initiation region. Primer pairs B2–B4 supported variability of transcriptional initiation within the canonical region.</p></div

Dispersed Human and Mouse Homeotic Loci at Which ESTs Were Detected on the Opposite Strand from the Homeotic Gene

<p>Loci with opposite-strand ESTs in both genomes are listed in the center box.</p