Rod TSS are conserved between human and mouse.

<p><b>A, B, C.</b> Combined genome-wide tracks of chromatin features in mouse retina (developmental changes in H3K4me2 occupancy (PN1, PN15 and RD1 PN30), CRX-binding (PN56), NRL-binding (PN28), developmental changes in DHS (1D, 1W, 8W)) and RNA-seq for human retina for genes TNFAIP3 (<b>A</b>), PLA2G5 (<b>B</b>) and CACNB2 (<b>C</b>) genes. Predicted rod TSS that shows conservation between mouse and human depicted as a black box.</p

Identification and prediction of alternative transcription start sites that generate rod photoreceptor-specific transcripts from ubiquitously expressed genes

<div><p>Transcriptome complexity is substantially increased by the use of multiple transcription start sites for a given gene. By utilizing a rod photoreceptor-specific chromatin signature, and the RefSeq database of established transcription start sites, we have identified essentially all known rod photoreceptor genes as well as a group of novel genes that have a high probability of being expressed in rod photoreceptors. Approximately half of these novel rod genes are transcribed into multiple mRNA and/or protein isoforms through alternative transcriptional start sites (ATSS), only one of which has a rod-specific epigenetic signature and gives rise to a rod transcript. This suggests that, during retina development, some genes use ATSS to regulate cell type and temporal specificity, effectively generating a rod transcript from otherwise ubiquitously expressed genes. Biological confirmation of the relationship between epigenetic signatures and gene expression, as well as comparison of our genome-wide chromatin signature maps with available data sets for retina, namely a ChIP-on-Chip study of Polymerase-II (Pol-II) binding sites, ChIP-Seq studies for NRL- and CRX- binding sites and DHS (University of Washington data, available on UCSC mouse Genome Browser as a part of ENCODE project) fully support our hypothesis and together accurately identify and predict an array of new rod transcripts. The same approach was used to identify a number of TSS that are not currently in RefSeq. Biological conformation of the use of some of these TSS suggests that this method will be valuable for exploring the range of transcriptional complexity in many tissues. Comparison of mouse and human genome-wide data indicates that most of these alternate TSS appear to be present in both species, indicating that our approach can be useful for identification of regulatory regions that might play a role in human retinal disease.</p></div

Rod TSSs are associated with PolII-binding sites in retina.

<p><b>A.</b> Number of TSS with different ratio of PolII-binding during development (comparison of PolII occupancy at PN25 and PN2; PN25-PN2) for rod-specific genes with single TSS (upper panel) and for rod (middle panel) or common (bottom panel) TSS of retina genes with multiple TSS. Blue–no changes; red–low PN25-PN2; green- high PN25-PN2. <b>B.</b> Developmental changes in PolII-binding (PN25-PN2 at TSS+/-1000bp) for rod and common TSS of retina genes with multiple TSS, compared with PolII-binding at TSS for control groups of genes (non-rod, cell-cycle and synapse). ***—p < 0.0001. <b>C.</b> Combined genome-wide tracks of chromatin features, as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig 1B</a></b> for <i>Kdm4c</i> gene. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene map. <b>D.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>C</b> for <i>Kdm4c</i> for mouse retina samples at PN1, PN15, adult and RD1 mutant. For comparison, normalized to <i>Gapdh</i> ΔCt values for each sample are in table below. Experiments done in duplicates with three technical replicas; ***—p < 0.0001.</p

Association of rod photoreceptor transcription factors CRX and NRL with rod TSS.

<p><b>A, C.</b> Number of TSS with different CRX (<b>A</b>) and NRL (<b>C</b>) binding for rod-specific genes with single TSS (upper panel) and for rod (middle panel) or common (bottom panel) TSS of retina genes with multiple TSS. Blue–no TF-binding; red–TF-binding. <b>B, D.</b> CRX (<b>B</b>) and NRL (<b>D</b>) accumulation at TSS (normalized number of reads at TSS+/-1000bp) for rod and common TSS of retina genes with multiple TSS, compared with control groups of genes (non-rod, cell-cycle and synapse). ***—p < 0.0001. <b>E, G.</b> Combined genome-wide tracks of chromatin features, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig <b>1B</b></a> for <i>Wdr17</i> (<b>E</b>) and <i>Tnfaip3</i> (<b>G</b>) genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene maps. <b>F, H.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>E and G</b> for <i>Wdr17</i> (<b>F</b>) and <i>Tnfaip3</i> (<b>H</b>) for mouse retina samples at PN1, PN15, adult and RD1 mutant, compare with mouse liver. For comparison, normalized to <i>Gapdh</i> delta Ct values for each sample are in table below. Experiments done in duplicates with three technical replicas; ***—p < 0.0001.</p

Epigenetic signature predicts employment of ATSS of ubiquitous gene in tissue-specific manner.

<p><b>A.</b> Pie chart presentation for number of TSS for different groups of rod-specific genes. <b>B.</b> All TSS for rod-specific genes were clustered based on chromatin signature around TSS+/-1000bp for following features: developmental changes in H3K4me2 occupancy (E17, PN1, PN7, PN15 and RD1 PN30), CRX-binding (PN56), NRL-binding (PN28), developmental changes in DHS (1D, 1W, 8W), developmental changes in PolII (PN2, PN25) as clustering criteria (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#sec002" target="_blank">methods</a> for details). <b>C, D.</b> Combined genome-wide tracks of chromatin features, as in <b>B</b> for <i>Gnb5</i> and <i>Rorb</i> genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box.</p

Rod TSSs are associated with DNase1 hypersensitive sites in retina.

<p><b>A.</b> Number of TSS with different ratio of DHS changes during retina development (comparison of DHS occupancy at PN56 and PN1; PN56/PN1) for rod genes with single TSS (upper panel) and for rod (middle panel) or common (bottom panel) TSS of retina genes with multiple TSS. Blue–no changes; red–low PN56/PN1; green- high PN56/PN1. <b>B.</b> Developmental changes in accessibility of different TSS by DHS (PN56/PN1 DHS at TSS+/-1000bp) for rod and common TSS of retina genes with multiple TSS, compared with control groups of genes (non-rod, cell-cycle and synapse). ***—p < 0.0001. <b>C.</b> Combine genome-wide tracks of chromatin features, as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig 1B</a></b> for <i>Tmem229b</i> gene. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene map. <b>D.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>C</b> for <i>Tmem229b</i> for mouse retina samples at PN1, PN15, adult and RD1 mutant, compare with mouse liver. For comparison, normalized to <i>Gapdh</i> ΔCt values for each sample are in table below. Experiments done in duplicates with three technical replicas; **—p < 0.001. <b>E, F.</b> Accessibility of different TSS by DHS (number of DHS at TSS+/-1000bp) during mouse retina (PN1, PN7, PN56) and brain (E14.5, E18.5, PN56) developments for rod (<b>F</b>) and common TSS (<b>E</b>) of retina genes with multiple TSS.*—p = 0.026 (<b>E</b>); ***—p = 0.0003 (<b>F</b>); *—p = 0.037/ 0.01.</p

Examples of the genes that use alternative transcription start sites in retina.

<p><b>A, C.</b> Combined genome-wide tracks of chromatin features, as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig 1B</a></b> for <i>Guk1</i> (<b>A</b>) and <i>Rtn4</i> (<b>C</b>) genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene maps. <b>B, D.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>A and C</b> for <i>Guk1</i> (<b>B</b>) and <i>Rtn4</i> (<b>F</b>) for mouse retina samples at PN1, PN15, adult and RD1 mutant, compare with mouse brain, liver and lung. For comparison, normalized to <i>Gapdh</i> delta Ct values for each sample are in table below. Experiments done in duplicates with three technical replicas; **—p <0.001; ***—p < 0.0001.</p

Use of alternative TSS for tissue specificity is universal.

<p><b>A-D.</b> Combined genome-wide tracks of H3K4me3 accumulation (UCSC tracks for mouse browser mm9) for different mouse tissue and cells (bone marrow, cerebellum, cortex, heart, kidney, liver, lung, mouse embryonic fibroblasts, spleen, thymus, bone marrow derived macrophages, murine erythroleukemia cells, olfactory bulb, placenta, small intestine and testis) for the new rod genes: <i>Kdm4c</i> (<b>A</b>) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g007" target="_blank">Fig 7C</a> for this gene); <i>Tnfaip3</i> (<b>B</b>) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g005" target="_blank">Fig 5H</a> for this gene); <i>Tmem229b</i> (<b>C</b>) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g006" target="_blank">Fig 6D</a> for this gene); <i>Ablim1</i>(<b>D</b>) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g003" target="_blank">Fig 3A</a> for this gene). In each case common TSS is depicted as a blue box, rod TSS–as a red box and other tissue-specific TSS–as green boxes. For reference on the top of each figure panel presents the genome-wide track of H3K4me2 for retina at PN15 and gene position and locus structure.</p

H3K4me2 epigenetic signature predicts tissue and cell specificity of TSS.

<p><b>A.</b> Changes of H3K4me2 occupancy around TSS+/-1000bp during mouse retina development as ratio of normalized number of reads at PN15/PN1 for rod and common TSS of retina genes with multiple TSS. At the bottom: average number of H3K4me2 reads accumulation for each group of TSS at PN1 and PN15. **p = 0.0011. <b>B.</b> Changes of H3K4me2 occupancy around TSS+/-1000bp during mouse erythropoiesis as ratio of normalized number of reads between late and early erythroblast for rod and common TSS of retina genes with multiple TSS. At the bottom: average number of H3K4me2 reads accumulation for both groups of TSS at early and late stages. <b>C.</b> Changes of H3K4me2 occupancy around TSS+/-1000bp during mouse brain development as ratio of normalized number of reads for whole brain and neuronal progenitor for rod and common TSS of retina genes with multiple TSS. At the bottom: average number of H3K4me2 reads accumulation for both groups of TSS at early and late stages. <b>D.</b> Changes of H3K4me2 occupancy around TSS+/-1000bp during mouse retina development for TSS of control groups of genes (non-rod, cell-cycle and synapse).</p

Confirmation of relationship between epigenetic signatures and gene expression.

<p><b>A, D.</b> Combined genome-wide tracks of chromatin features, as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig 1B</a></b> for <i>Ablim1</i> (<b>A</b>) and <i>Usp6nl</i> (<b>D</b>) genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene maps. <b>B, E.</b> Patterns of <i>Ablim1</i> and <i>Usp6nl</i> genes expression during mouse retina development; reanalyzing of microarray data from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.ref016" target="_blank">16</a>]. <b>C, F.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>A and D</b> for <i>Ablim1</i> (<b>C</b>) and <i>Usp6nl</i> (<b>F</b>) for mouse retina samples at PN1, PN15, adult and RD1 mutant, compare with mouse brain and spleen. For comparison, normalized to <i>Gapdh</i> delta Ct values for each sample are in table below. Experiments done in duplicates with three technical replicas; ***—p < 0.0001.</p