Complex Loci in Human and Mouse Genomes

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis–antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis–antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis–antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes

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

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

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

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

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

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

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

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to &lt; 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of &amp; GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P &lt; 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo