Human promoter CTAG1A and modified constructs.

<p>(<b>a</b>) The 535 base pair long promoter region of human gene CTAG1A is rich in CpGs and exhibits α-scores higher than the genomic distribution with pronounced peaks. Shown are the composite α_k-scores (top), the individual α_k-scores for different sizes of <i>k</i> in the middle graph (colour coded, blue = negative, red/orange = positive), and CpGs in yellow (bottom). The three strongest regions are marked by red bars. (<b>b</b>) In-vitro activity of the original CTAG1A promoter (hCTAG1A Promoter), the three strongest α-score regions deleted (hCTAG1A delta), the three strongest α-score regions replaced with sequences from the genomic concatomer (hCTAG1A replace), and the three strongest α-score regions replaced with sequences from the promoter-like concatomer (hCTAG1A UP). Also shown are results without any promoter (Negative CO) and the SV40 core promoter (SV40 Promoter AVG).</p

Calculation of the binding constants <i>k</i>_on, <i>k</i>_off, and <i>K_D</i> for the TFIIB binding assays.

<p>Calculation of the binding constants <i>k</i>_on, <i>k</i>_off, and <i>K_D</i> for the TFIIB binding assays.</p

<i>In-vitro</i> promoter activity driven by artificial constructs.

<p>Artificial constructs ArS110, ArS300, ArS201 and ArS232 exhibit strong promoter activity driving a reporter gene (firefly luciferase, internally normalized by renilla luciferase) in mammalian cell lines: (<b>a</b>) CHO/hamster, (<b>b</b>) P19/mouse, (<b>c</b>) VERO/monkey, (<b>d</b>) HEK293/human, but not in (<b>e</b>) the insect cell line Sf9/army worm. Also shown are the negative control (−) and the SV40 core promoter activity (+). (<b>f</b>) TATA-boxes 1 (left) and 2 (right) were deleted from construct ArS232: deletion of TATA-box 1 only (dT1) results in lack of activity, deletion of TATA-box 2 (dT2) does not change expression levels, while deletion of both (dT1&2) results in slightly increased expression levels.</p

Binding affinity of artificial promoter constructs to the transcription factors TFIIB and TBP.

<p>The binding expressed as Δnm on the y-axis was monitored in real time as sec (x-axis), using the ForteBio Octet QK instrument. Binding was conducted in four phases: (i) loading of biotinylated DNA fragments to the streptavidin biosensor tip, (ii) washing in Kinetics Buffer, (iii) association of the transcription factor and (iii) dissociation of the transcription factor. (<b>a</b>) The promoter constructs ArS110, ArS201, ArS232 and ArS300 show similar binding affinities to the TFIIB protein. (<b>b</b>) The promoter constructs ArS232, ArS232 dT1, ArS232 dT2 and ArS232 dT12 exhibit sequence-specific binding to the TBP protein. ArS232 dT12 lacking two TATA-Boxes shows the lowest binding affinity compared to the other constructs. (<b>c</b>) TFIIB binding vs. a negative control, for which we chose a 85 bp long sequence from inside the coding region of the luciferase gene (pGL3-Basic Promoter Promega: 1314 bp–1399 bp).</p

Comparison of canFam2.0 and canFam3.1.

<p>Comparison of canFam2.0 and canFam3.1.</p

Location of lincRNAs and single-exon intergenic non-coding transcripts.

<p>(a) We show the mapping of lincRNAs, broken down by sample across chromosome 1. (b) Histogram of distance from intergenic transcripts to the next known transcribed element in both the 5′ (left) and 3′ (right) direction. The average distance is around 150,000 nucleotides, suggesting that these loci are not closely associated with known genes.</p

Transcribed loci per tissue and library preparation.

<p>N/A, due to poor alignment performance, this library was excluded from subsequent analyses.</p

Distance trees of expression profiles.

<p>We constructed neighbor-joining trees based on the correlation between expression values (FPKM>1.0) between samples, with 1 minus Spearman's rho defining the distance. Colors denote library construction methods (poly-A: blue, DSN: red). We divided transcribed loci into (a) protein coding genes with RNA-Seq support, either annotated by EnsEMBL in dog or EnsEMBL in the human orthologous regions. Replicates cluster together, so do the library constructions methods poly-A and DSN, as well as related tissues, such as heart and muscle; (b) antisense transcripts, that overlap at least one exon of a protein coding gene, as defined in (a). With the exception of testis, poly-A and DSN separate the samples, with both the poly-A and DSN sub-trees maintaining closer relationships between the related tissues heart and muscle; (c) spliced intergenic loci, excluding sequences that have coding potential. Similar to protein coding genes, the poly-A and DSN group by tissue first, with the exception of kidney DSN; and (d) intergenic and uncharacterized single-exon transcript loci. In this set, DSN and poly-A are, similar to antisense loci, the most dominant factor when grouping samples.</p