Conservation in first introns is positively associated with the number of exons within genes and the presence of regulatory epigenetic signals
© Park et al.; licensee BioMed Central Ltd. 2014
Received: 7 January 2014
Accepted: 18 June 2014
Published: 26 June 2014
Genomes of higher eukaryotes have surprisingly long first introns and in some cases, the first introns have been shown to have higher conservation relative to other introns. However, the functional relevance of conserved regions in the first introns is poorly understood. Leveraging the recent ENCODE data, here we assess potential regulatory roles of conserved regions in the first intron of human genes.
We first show that relative to other downstream introns, the first introns are enriched for blocks of highly conserved sequences. We also found that the first introns are enriched for several chromatin marks indicative of active regulatory regions and this enrichment of regulatory marks is correlated with enrichment of conserved blocks in the first intron; the enrichments of conservation and regulatory marks in first intron are not entirely explained by a general, albeit variable, bias for certain marks toward the 5’ end of introns. Interestingly, conservation as well as proportions of active regulatory chromatin marks in the first intron of a gene correlates positively with the numbers of exons in the gene but the correlation is significantly weakened in second introns and negligible beyond the second intron. The first intron conservation is also positively correlated with the gene’s expression level in several human tissues. Finally, a gene-wise analysis shows significant enrichments of active chromatin marks in conserved regions of first introns, relative to the conserved regions in other introns of the same gene.
Taken together, our analyses strongly suggest that first introns are enriched for active transcriptional regulatory signals under purifying selection.
Recent complete sequencing projects have confirmed that almost all eukaryotes have introns [1–6]. Different species harbor dramatically different density and length of introns, ranging from a few bps to hundreds kbps [5, 7, 8]. Generally speaking, genes in higher eukaryotes such as mammals have a greater number of introns than those of lower eukaryotes such as yeast, Drosophila, and C. elegans[5, 7, 8]. These differences may partly be explained by the differences in modes of intron removal between lower and higher eukaryotes , as well as differences in selective pressure. A substantial fraction of introns in the human genome have likely originated early in eukaryotic evolution, and dynamic evolutionary changes such as intron gain and loss have structured the eukaryotic introns since [10–14]. Noticeably, the number of genes is relatively stable across organisms from C. elegans (~19,000) and Drosophila (~14,000) to humans (~25,000), while the fraction of non-coding DNA including introns greatly varies up to several folds [15, 16], some of which is likely to underlie species-specific adaptations [17, 18].
The mere existence of introns in the eukaryotic genome is intriguing, given the cost of transcription and the cost of maintaining a splicing regulatory system that ultimately eliminates the introns from the functional product of the gene. In particular, whether introns have evolved under selective constraints, and the extent thereof, are not entirely clear; while some studies suggest that introns evolve largely free from selective constraints [19–22], others imply that intron sequences are subject to considerable levels of evolutionary constraints [22–25]. Using intronic multispecies conserved sequences (MCSs), Sironi et al.  showed that the MCS density steadily increase with intron length with MCSs occupying up to 10% of total size in long introns, and also that MCSs are enriched in genes involved in development and transcription. Based on 225 intron fragments in D. melanogaster and D. simulans, Haddrill et al.  demonstrated that a substantial portion of intronic sites is likely to be evolving under considerable selective constraint and this tendency increases with intron length. Furthermore, Vinogradov  showed that the length of conserved intronic sequence in the human genome is greater in proteins with the larger numbers of functional domains. Even though several reports have shown an enrichment of conserved sequences in introns, in various species [23–25, 28, 29], these claims are not without controversies. In one instance, it was shown that intronic sequences evolve faster than fourfold degenerate sites when splicing regulatory sequences were excluded . These discrepancies can be partly ascribed to biases in the data sets with different ranges of lengths of introns studied . Besides their obvious role in isoform regulation, introns have also been shown to harbor regulatory signals and noncoding genes [30–33]. Thus, overall, it is highly likely that portions of intronic sequences are evolving under selective constraints consistent with their functional importance.
The 5’-most “first” intron differs from the other introns in several aspects in terms of processing, epigenomic marks, length and evolution. First, in terms of processing, despite an overall 5’-to-3’ trend in splicing during transcription, the first intron is removed (on average) somewhat later . Second, the 5’ end of the first intron displays a specific epigenomic context being enriched for two activating histone modifications H3K4me3 and H3K9ac . Finally, the “first” introns have a special status, as these are typically the longest among all introns and appear to be most selectively constrained. More conserved blocks were found in first introns relative to other introns in several species . Consistently, some studies have shown that intron divergence has a significant negative correlation with the length of first introns in Drosophila[27, 37]. Zheng et al.  have also reported that, in Tetrahymena, the most conserved introns are found closer to the 5’ end of genes. Furthermore, introns harboring regulatory elements tend to be the first introns [38–40], and, in fact, the frequencies of certain regulatory motifs are greater in first introns . Overall, it seems that, among the various possible roles of introns, first introns have especially evolved to harbor regulatory elements.
List of data resources
UCSC chromosome sequence
46 species multiple alignment
The Genome Reference Consortium
DNaseI Hypersensitivity Uniform Peaks from ENCODE/Analysis
Transcription Factor ChIP-seq Uniform Peaks from ENCODE/Analysis
Histone Modifications by ChIP-seq from ENCODE/Broad Institute
First introns are the most conserved
As a proxy for purifying selection, we compared evolutionary conservation across introns grouped by their position in the transcript structure. Conservation in an intron was estimated as the fraction of intron sites that were conserved based on a PhastCons score threshold. Three different multiple alignments (primates, mammals and vertebrates) and different PhastCons score thresholds were used. Our conclusions hold for all choices of alignment and threshold; here we only present the results based on mammalian conservation with PhastCons score threshold of 0.5. The primary focus of our investigation was transcriptional regulatory elements in introns. Therefore, to exclude the possibility of splice site signals biasing the conservation score, especially for short introns, the sequences within 300 bps of the splice junction, which are considered to harbor splicing regulation signals , were excluded from our analysis. Based on these criteria, the median conservation per intron was only 2.1%. Introns were grouped by their positions from the 5’ end of the transcript; for instance, all first introns were in the first group. The fraction of conserved sites was then estimated within each group.
Chromatin signals are the highest in first introns and increase with increasing conservation
Conserved regions in first intron are likely to play a transcriptional regulatory role as suggested by previous studies [43–51]. Several chromatin marks have been shown to associate with transcriptional regulatory regions [52–54]. Next we assessed whether the first introns are enriched for specific regulatory signals, similar to the conservation analysis. We obtained a number of chromatin marks and protein-binding data from ENCODE [55, 56] for three cell lines - GM12878, H1-hESC, and K562. The following data were included: DNase I hypersensitivity sites (DHS), Transcription factor binding sites (TFBSs) for 80 TFs in GM12878, 50 TFs in H1-hESC, and 112 TFs in K562, active chromatin marks (e.g., H3K4me1, H3K4me3), a repressive chromatin mark (e.g., H3K9me3), and the insulator protein CTCF binding sites.
Next we contrasted the above conservation results for first introns with that for proximal promoters of the genes, known to harbor conserved regulatory signals [57–61]. We found that the correlations between conservation and epigenomic marks also hold for 2 kb proximal promoter regions of the gene (Additional file 1: Figure S4A-C), which suggests an enrichment of conserved regulatory signals specifically in first introns, akin to proximal promoters.
First intron conservation and regulatory signals positively correlate with the numbers of exons
Conservation and active epigenomic marks correlate with level of gene expression
Although the analysis thus far was based on all ‘transcripts’, we repeated the analyses by selecting a single representative transcript per gene, resulting in ~16,000 transcripts. As shown in Additional file 1: Figure S7A-D, the analyses at the gene level yield qualitatively the same results.
Regulatory signals preferentially occur in conserved regions in first introns - a gene-wise assessment
Trends in the first introns are independent from those in promoters
A substantial portion of transcriptional control of a gene is mediated by signals in its proximal promoters. In fact, numerous ChIP-seq data for TFBS from the ENCODE project reveal a distribution of binding sites around the transcription start site, into the introns [56, 64]. A 5’ enrichment of certain epigenetic marks has also been noted with regards to splicing signals .
We first investigated whether the proportions of conservation and epigenetic marks are biased toward the 5’ end of the intron. After excluding the short first introns (shorter than the median length) each intron was binned into five equal-sized bins, and we estimated for each bin, the fraction of introns in which the highest signal in the intron was in the particular bin. As shown in Additional file 1: Figure S9, there is varying enrichment of signals toward the 5’ bins but in absolute terms only in a small fraction of introns the highest signal is in the 5’-most bin. Moreover, this trend is not uniformly observed for all regulatory signals.
Despite an enrichment of signals toward 5’ as well as the fact that the general patterns of enrichment in the first introns also hold for the 5’ flanking regions (Additional file 1: Figure S4 and S5), it is not immediately clear whether the patterns in the two regions are related by virtue of extended conserved and regulatory regions spanning the promoter and the first introns. To ensure that the observed patterns in first introns are not simply due to signals spanning the two regions, we tested if the trends in the first introns are maintained after removing such “spillover” signals. We reasoned that the genes in which a particular signal in first intron is simply due to spillover from the promoter should exhibit a greater proportion of that signal in promoter and first exon relative to the first intron. Thus we excluded the genes in which the proportion of a signal in the first exon and promoter was at least as high as that in the first intron. Despite the reduction in statistical power owing to much reduced dataset, all the trends were still maintained (Additional file 1: Figure S10A-D). Next, to exclude the interference of signals between introns and exons or flanking regions of other genes, we repeated the analyses after excluding the genes whose first intron overlapped with exon or flanks of another gene. As shown in Additional file 1: Figure S11A-D, all trends in the first introns are still maintained after removing the overlapped sets. All these results suggest that the first intron conservations and the enrichment of regulatory signals are independent of the trends for the promoter.
Trends in the first introns are not due to their proximity to the transcriptional start site
Given that many of the trends in first intron are similar to those in the proximal promoter, next we assessed whether the trends in the first introns are simply due to their proximity to the transcription start site (TSS). Thus we investigated the trends in the first introns compared to that in the second introns when controlled for their distance from the TSS. We categorized first and second introns into bins corresponding to distance of the 5’ end of the intron from the TSS and compared the first and the second intron trends within each bin. Note that because first introns are much longer than the second intron (Additional file 1: Figure S12A) and because we are controlling for the distance to the 5’ end of the intron, in the direct comparison, on average, the distances within first introns is much greater than those in second introns, thus rendering this comparison conservative. As shown in Additional file 1: Figure S12A, distances from the TSS to the first and the second intron are vastly different and overlap only in a short range 500–1000 bps. For various distance bins within in this range, we compared the conservation and chromatin signal proportions between the first intron and the second intron.
Introns are ultimately removed from protein synthesis as a part of post-transcriptional processing, yet all the intron sequences are respectively copied as pre-mRNAs or DNAs sequence-by-sequence during transcription and replication, which seems to cause huge energetic burden to cell. About 2 ATP is known to be consumed for 1 bp synthesis during transcription . Considering that introns can be as long as hundreds of kilobases, their maintenance must entail a substantial cost to the cells. Nevertheless, most eukaryotic genes have introns [1–6], and although introns can be lost, in general, introns have been maintained during eukaryotic lineage evolution [5, 6, 66, 67]. Evolutionary maintenance of introns despite the energetic burden they entail suggests some evolutionary advantage afforded by introns.
Several studies have presented various possible scenarios for how introns provide advantage to cell’s survival [68–72]. It is also true that, for a long time, introns have been considered to be non-essential for the most part [19–21]. In fact, approximately 3% of genes in the human genome are intronless genes . Furthermore, no significant functional changes have been detected in many experimental designs with or without introns for the same coding sequences ; however this counterintuitive finding may be simply due to the fact that in molecular biology experiments, ‘gene function’ generally has been equated to ‘protein function’, devoid of its regulatory context.
Transcriptional regulatory signals encoded within introns represent one of the main selective advantages afforded by introns . Two types of regulatory signals have been reported in introns; classical enhancers and intron-mediated enhancers (IME). Several classical enhancers, i.e., cis-regulatory elements regulating spatio- temporal gene expression, are known to locate within introns in mouse transgenic experiments , for instance, the enhancer elements of GLI3, an important transcription factor of Sonic hedgehog signaling . In contrast, IME suggest a broad role of an intron, first intron in particular, in regulating expression level, without ascribing the function to a specific region within the intron, suggesting a mechanism different from that for classical enhancers [32, 77]. For instance, in experiments performed in Arabidopsis, rice, as well as mammal, the expression level of a gene with intron, particularly first intron, could increase up to 100-fold compared to the expression construct with the same coding sequences but without the introns . IME activity was found to be dependent on the location and distance from transcription start site unlike the classical enhancers . Whether or not IME is a general mechanism of expression control is not known.
Here, we have investigated the functional importance of first introns in human by quantifying their evolutionary conservation and potential regulatory content relative to other introns. Sequence conservation is considered to reflect the resistance against random mutations through purifying selection. Identifying conserved regions in genomes has thus been one of primary criterion to detect functional regions of genomes. Previous studies in several species including Drosophila and Arabidopsis[27, 37, 41, 80] have shown that first introns tend to be the longest and the most conserved, which is recapitulated in human by our study (Figure 1). We further investigated the reasons of higher conservations in first introns by testing their association with various regulatory marks, and the associations of conservation and regulatory signals in first introns with the gene’s expression level (Figures 2, 3, 4, 5 and 6). This analysis also underscores the importance of epigenomic data, which became available only recently by ENCODE project (Table 1), in interpreting the function of the non-coding portion of the genome. One of the interesting findings of this work is that genes with higher density of conservation and active regulatory marks but not repressive marks in first introns tend to have more exons that encode longer proteins (Figures 4 and 5), which can be interpreted to suggest that long functionally complex proteins may also be under a richer regulatory control. It is not entirely clear why only active regulatory signals but not repressive signals have positive correlations with conservation and number of exons. It may partly be due to the tendency of repressive signals to be broad and less intense relative to activating signals , which can result in lower discrimination of a specific region as well as lower detectability and lower statistical power. Overall, by leveraging the recent explosion in epigenomic data, our work lends further support, particularly in human, to the notion that introns, and especially the first introns, harbor evolutionarily constrained regulatory regions mediating both the level and complexity of gene expression. However several important questions remain open.
In our gene-specific analysis of enrichment of various regulatory signals in the conserved portion of the first intron, we found that in first introns, the conserved regions are favored by DHS, TFBS binding, and H3K4me3, which suggests that the conserved region may have a role in active gene regulation. More interestingly, we found a difference in two activating marks – H3K4me1 and H3K4me3. While both these marks are associated with proximal promoters, only H3K4me1 is associated with distal enhancers . This subtle difference in enrichment may suggest that the conserved regions in the first intron are more promoter-like and less like a distal enhancer in their mode of action. However, this effect is not simply due to spillover of promoter signals into enhancer as we showed above (Additional file 1: Figure S10A-D). Further mechanistic disambiguation of this difference will require additional studies.
In the present study, we investigated the potential regulatory role of first introns in human genes by leveraging the recent explosion in epigenomic data by the ENCODE project. In addition to extending the previous results in Drosophila and plant to human, i.e. showing that the first introns are enriched for conserved regions, we show that these higher conservations in first introns are related to 1) the presence of active regulatory chromatin marks, 2) higher expression levels of genes, and 3) a greater number of exons within genes. Overall, our results strongly suggest that first introns in human are enriched for evolutionarily selected active transcriptional regulatory signals that are likely to be important for regulating complex gene expression patterns of large multi-domain genes.
The precise mechanism by which individual conserved, putative regulatory regions in the first intron, regulate gene expression, as well as other potential functions of conserved regions in the first intron, are unclear. The extreme lengths of mammalian first introns represent another enigma. The evolutionary path leading up to long introns as well as whether the first intron length is under selective constraint are not known. Finally, whether it is beneficial for the regulatory elements to reside within the first intron, as opposed to, say, the upstream region of the gene, or whether evolution is neutral to this outside-inside choice, is another open and interesting question. To ultimately resolve the mystery of introns, these are some of the questions that will need to be addressed.
Obtaining of introns, 5’-, and 3’-flanking regions from the human genome
The exon-intron position information of 36,024 Refseq mRNA (KnownGenes) without duplicates was downloaded from the UCSC Table Browser (January 2013). Genomic sequences for each chromosome were obtained from the primary GRCh37/hg19 assembly, and were used for retrieve intron sequences. Very short introns (less the 1 kb in length) were excluded, as well as very long introns (greater than (third quartile + (interquartile range × 1.5)) in length to minimize the outlier effects. To minimize interference from splicing signals in interpreting intronic conservation, we excluded 300 bps from both the 5’ and 3’ ends of each intron . Then repeat elements were masked by RepeatMasker . The numbers for introns in each ordinal position group after all these filtrations are shown in Figure 1. Additionally, we obtained the 2 kb region upstream of the first exon and 2 kb region downstream of the last exon, as a proxy for 5’ and 3’ flanks of genes.
PhastCons scores were used to estimate position-wise sequence conservation . The PhastCons scores for 46 placental mammal subset were downloaded from the UCSC genome browser (Table 1, March 2013). The Phastcons scores were overlaid onto the intron regions and 5’/3’-flanking regions obtained by the methods described above section. Sites with PhastCons score ≥ 0.5 were considered as ‘Conserved’ sites. The proportions of conserved sites were then estimated for each group of introns grouped by their ordinal positions, and 5’/3’-flanking regions. Conservation in first intron for each gene was estimated by the number of conserved sites divided by the total length of the intron.
Obtaining and mapping regulatory signals and chromatin marks
Genome positions of peaks (region of statistically significant signal enrichment) for DHSs, TFBSs and chromatin marks measured in the ENCODE Tier-1 cell lines (GM12878, H1-hESC and K562) were downloaded from the ENCODE Project from UCSC genome browser (March 2013). The specific download links are provided in Table 1. The processed peak regions of all of these regulatory signals were extracted, and mapped onto the filtered introns and flanking regions. For each intron and flanking region, we then estimated the proportion of the region covered by each regulatory signal and chromatin mark.
Obtaining mRNA expression levels estimated by RNA-seq analysis
Krupp et al.  have reported the mRNA expression levels in RPKM. We used Log2 (RPKM + 1) to report expression levels of mRNAs for 11 different human tissues (Table 1). A total of 32,384 mRNAs have their expression values.
Kendall’s tau rank correlation coefficients were computed using R studio (Racine 2012) among the variables representing evolutionary conservations of introns or flanking regions, regulatory signals mapped onto introns or flanking regions, and mRNA expression levels. Introns or flanking regions with no conservation or regulatory signal were excluded; we ascertained that this only makes our results more stringent because the fraction of first introns that were eliminated were smaller than those for other introns and including this with “0” value would make the results even stronger.
To test whether the regulatory signals and chromatin marks are enriched in conserved regions compared to non-conserved regions in each gene, odds ratio of the signal estimates between the two regions was estimated. Briefly, first introns were divided into two regions, conserved first introns and non-conserved first introns by the criteria of Phastcons ≥ 0.5, and the chromatin signals and regulatory signals described in the main text were overlaid into each region, generating a 2×2 contingency table. Odds ratios were then estimated for each gene from the contingency table. The odds ratio is the ratio of the proportion of regulatory signals in conserved sequences to that in non-conserved sequences. The odds ratios were thus computed using the “Text::NSP::Neasures::2D::odds” Perl module, and the 95% confidence intervals associated with odds ratios were calculated using the formulas, (SC: Signal peaks on conserved sites, NC: Non-signals peaks on conserved sites, SN: Signal peaks on non-conserved sites, NN: Non-signal peaks on non-conserved sites) .
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A1A4A01003793) and by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0016) to S.S.C, and by NIH grant GM100335 to S.H. Authors would like to thank Steve Mount, Srutii Sarda, and Avinash Das for their useful comments.
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