Tracks through the genome to physiological events

Abstract

What is the topic of this review? We discuss tools available to access genome-wide data sets that harbour cell-specific, brain region-specific and tissue-specific information on exon usage for several species, including humans. In this Review, we demonstrate how to access this information in genome databases and its enormous value to physiology. What advances does it highlight? The sheer scale of protein diversity that is possible from complex genes, including those that encode voltage-gated ion channels, is vast. But this choice is critical for a complete understanding of protein function in the most physiologically relevant context. Many proteins of great interest to physiologists and neuroscientists are structurally complex and located in specialized subcellular domains, such as neuronal synapses and transverse tubules of muscle. Genes that encode these critical signalling molecules (receptors, ion channels, transporters, enzymes, cell adhesion molecules, cell-cell interaction proteins and cytoskeletal proteins) are similarly complex. Typically, these genes are large; human Dystrophin (DMD) encodes a cytoskeletal protein of muscle and it is the largest naturally occurring gene at a staggering 2.3 Mb. Large genes contain many non-coding introns and coding exons; human Titin (TTN), which encodes a protein essential for the assembly and functioning of vertebrate striated muscles, has over 350 exons and consequently has an enormous capacity to generate different forms of Titin mRNAs that have unique exon combinations. Functional and pharmacological differences among protein isoforms originating from the same gene may be subtle but nonetheless of critical physiological significance. Standard functional, immunological and pharmacological approaches, so useful for characterizing proteins encoded by different genes, typically fail to discriminate among splice isoforms of individual genes. Tools are now available to access genome-wide data sets that harbour cell-specific, brain region-specific and tissue-specific information on exon usage for several species, including humans. In this Review, we demonstrate how to access this information in genome databases and its enormous value to physiology.

Figure 1. Visualizing various tracks using UCSC Genome Browser reveals the location of four alternatively spliced exons of mouse Cacna1b based on mouse genome version Dec. 2011 (GRCm38/mm10; http://genome.ucsc.edu/cgi‐bin/hgGateway; Kent et al. 2002)

The genomic regions flanking alternatively spliced exons (18a, 24a, 31a and 37a/37b) of Cacna1b are shown in A–D. In each panel, scale bars indicate the size of the genomic region shown. Horizontal layers I–IV represent different display options, as follows: layer I, possible splice patterns in the region of Cacna1b; and layers II–V, tracks of UCSC Genes (blue), Ensemble Genes (maroon), and two different display options for Vertebrate Conservation (grey and green). Exons are shown as rectangles (layer I), and the direction of transcription is indicated by small arrows (layer III). The conservation track (layers IV and V) displays the PhastCon scores (from 0 to 1) calculated based on the genome sequence alignment of 60 vertebrates. In each panel, an alternatively spliced exon is captured in at least one Ensemble transcript. Not all alternatively spliced exons appear in mm10 UCSC Genes (or Refseq Genes track, not shown), but they align perfectly with peaks of highest conservation in the Vertebrate Conservation track. The following steps will recreate the display shown. (i) In the UCSC Genome Browser, choose ‘Genomes’ top left, group ‘mammal’, genome ‘mouse’, assembly ‘GRCm38/mm10’, search term ‘Cacna1b’, location chr2:24603889‐24763152, submit. (ii) Scroll down to ‘Genes and Genes Predictions’ header category, set ‘Ensembl genes’ tab to ‘full’ and ‘UCSC Genes’ tabs to ‘dense’ and set all other tabs to ‘hide’. (iii) Scroll down to Comparative Genomics header, set ‘Conservation’ to display ‘full’ and click on the ‘Conservation’ link; here you can select subtracks by clade, select ‘phastCons’ scores in ‘full’, and ‘hide’ other features, further restrict the PhastCons scores to ‘60 Vert. Cons’ in the subtrack lists, ‘submit’. The complete Cacna1b gene is displayed. A–D are zoomed in to resolve regions that contain alternatively spliced exons. To recreate each panel type in locations: chr2:24678405‐24686581 (18a region; A); chr2:24653647‐24657925 (24a region; B); chr2:24638102‐24649580 (31a region; C); and chr2:24621473‐24632972 (37 region; D). Co‐ordinates of exons are as follows: chr2:24682976‐24683038 (18a); chr2:24656711‐24656722 (24a); chr2:24642853‐24642858 (e31a); chr2:24626823‐24626919 (37a); and chr2:24625238‐24625334 (37b). Alternatively spliced exons are flanked by consensus dinucleodide splice junction motifs AG and GT. Functions and tissue‐specific distributions of these alternative exons have been described by our laboratory (Lipscombe et al. 2013 a).

Figure 2. The use of different first exons in mouse Cacna1c in heart and brain is readily visualized using the UCSC Genome Browser by viewing H3K4Me3 regulatory markers that often correlate with the sites of transcription initiation

Mouse July 2007 (NCBI37/mm9) assembly is used for display, with genomic co‐ordinate chr6:118534231‐119182730. Genomic location and scale bar indicate the position and coverage of the genome. Each line represents a reference sequence aligned to the mouse genome (mm9) from UCSC Genes track (blue). Tissue‐specific histone modifications are displayed in the ChIP‐seq subtrack from the ENCODE/Ludwig Institute for Cancer Research (LICR; ENCODE, 2012) in parallel with RNA‐seq data from mouse heart (8 weeks) and mouse embryonic whole brain (day 14.5). Exon 1a is used in heart and is located > 80 kb upstream of exon 1b that is used in both heart and brain. Three H3K4Me peaks are shown that correspond to two different transcription start sites for Cacna1c and a single transcription start for Dcp1b. The Dcp1b gene is transcribed on the reverse strand.

Figure 3. Visualization of tissue‐specific use of alternative promoters in human CACNA1C using the integrative genomics viewer (IGV; Thorvaldsdóttir et al. 2013)

The 2009 human (GRCh37/hg19) assembly was used in this figure to visualize the two alternative promoters in CACNA1C. Junction Box option is turned on for quantitative visualization of exon–exon junction from mRNA sequencing reads aligned to gene annotations. These plots show tissue‐specific usage of exon 1a (heart) and exon 1b (heart and brain). The thickness of the connections indicates the frequency of the reads that connect exon–exon junctions. The sense strand (red) and antisense strand (blue) are shown. Data were loaded from ‘Bodymap 2.0’ from IGV. The DPC1B gene is short and transcribed in the reverse direction. There are two promoters in CACNA1C, marked 1a and 1b.

Figure 4. Tissue‐specific use of alternative exons in human CACNA1C visualized using the GTEx portal (GTEx, 2013)

Using the GTEx portal, the major exons and patterns of alternative splicing are displayed. The blue rectangles represent exons and the red circles the splice options. The colour intensity represents the frequency of sequence reads for the specified tissue. Shown are analyses of RNA‐seq data from human coronary artery and heart ventricles. The majority of Ca_V1.2 expressed sequence in heart differs from that expressed in coronary artery at two mutually exclusive sites. In heart, exons 1a and 8a are expressed, whereas in coronary artery exons 1b and 8b are expressed.