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Alternative splicing regulated by butyrate in bovine epithelial cells.

Wu S, Li C, Huang W, Li W, Li RW - PLoS ONE (2012)

Bottom Line: This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing.Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control.Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Center for Research in Biological Systems, University of California San Diego, San Diego, California, United States of America.

ABSTRACT
As a signaling molecule and an inhibitor of histone deacetylases (HDACs), butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control (CT) groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001) at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor) and Exon#11 (Acceptor) in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

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Relative abundance of transcript isoforms of aminoacylase 1 gene.Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Blue: butyrate-treated cells; Red: untreated control cells. Y-axis: normalized read counts. ***indicates P<0.0001.
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pone-0039182-g003: Relative abundance of transcript isoforms of aminoacylase 1 gene.Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Blue: butyrate-treated cells; Red: untreated control cells. Y-axis: normalized read counts. ***indicates P<0.0001.

Mentions: Mixture-of-Isoforms algorithm (MISO), a probabilistic framework that enables quantification of the expression level of alternatively spliced genes and identification of differentially regulated isoforms from RNA-Seq data [18], was used to detect differentially expressed isoforms induced by butyrate in bovine epithelial cells. In this study, MISO detected a total of 216 isoforms from 98 genes that were differentially regulated by butyrate using a combined cutoff of ΔΨ >0.20 (i.e., the posterior distribution over the change in Psi for each event) and Bayes factor >10. The Bayes factor represents the weight of the evidence in the data in favor of differential expression. For example, Bayes factor =2 means that the isoform is two times more likely to be differentially expressed. Of these events, 50 isoforms from 36 genes were supported by at least 3 pair-wise comparisons (Table 4); 29 of the 36 genes had two mRNA isoforms while the rest had at least 3 isoforms. The mRNA-level expression of defensin, beta 1 gene (DEFB1) was not detected in control cells but significantly up-regulated by butyrate (FDR <0.001). This gene had two isoforms. Isoform #1 had 3 exons and was not detectable in control cells but barely detectable in butyrate-treated cells. However, Isoform#2 had 2 exons and was strongly induced by butyrate (Fig. 1). Similarly, IL-18 (ENSBTAG00000000277) had 2 isoforms with the same number of exons. These 2 isoforms differed in the exon structure, nevertheless. Isoform#1 had a similar number of assigned sequence reads while Isoform#2 had approximately 10 times more assigned reads in butyrate-treated cells (252.08±30.51, mean ± sd) than in control cells (25.94±13.69) (P<0.0001, Fig. 2). Isoform #1 of origin recognition complex, subunit 1 (ORC1, transcript# ENSBTAT00000052505) had mean read counts of 168.26±52.75 (± sd) and 1239.75±48.02 in butyrate and control groups, respectively, and was strongly repressed by butyrate. Isoform#2 of ORC1 remained unchanged. Histone deacetylase 10 (HDAC10) had 2 isoforms with the read count ratio between dominant (ENSBTAT00000014602) and minor (ENSBTAT00000042583) isoforms greater than 150 in the control cells. Similarly, the dominant isoform was significantly repressed (P<0.00001), while the minor isoform was not affected by butyrate. However, the expression of both isoforms of HDAC5 was strongly enhanced by butyrate treatment (data not shown). For genes with ≥3 isoforms, one or 2 isoforms were often differentially regulated by butyrate. As Figure 3 shows, the expression level of Isoforms# 1 and 3 of aminoacylase 1gene (ACY1) was unchanged but that of Isoform#2 was strongly repressed by butyrate (Fig. 3). On the other hand, Isoform#2 of coiled-coil domain containing 24 (CCDC24) was little changed in response to butyrate treatment while Isoforms#1 and 3 were significantly induced by butyrate (Fig. 4). Moreover, differential expression of 6 selected transcript isoforms induced by butyrate, including transcripts such as ENSBTAT00000009005 (LGALS9), ENSBTAT00000010687 (CLSTN3), ENSBTAT00000012035 (MX1), ENSBTAT00000020401 (NASP), ENSBTAT00000036545 (GUK1), and ENSBTAT00000061158 (TACC2), were confirmed using RT-PCR.


Alternative splicing regulated by butyrate in bovine epithelial cells.

Wu S, Li C, Huang W, Li W, Li RW - PLoS ONE (2012)

Relative abundance of transcript isoforms of aminoacylase 1 gene.Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Blue: butyrate-treated cells; Red: untreated control cells. Y-axis: normalized read counts. ***indicates P<0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375255&req=5

pone-0039182-g003: Relative abundance of transcript isoforms of aminoacylase 1 gene.Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Blue: butyrate-treated cells; Red: untreated control cells. Y-axis: normalized read counts. ***indicates P<0.0001.
Mentions: Mixture-of-Isoforms algorithm (MISO), a probabilistic framework that enables quantification of the expression level of alternatively spliced genes and identification of differentially regulated isoforms from RNA-Seq data [18], was used to detect differentially expressed isoforms induced by butyrate in bovine epithelial cells. In this study, MISO detected a total of 216 isoforms from 98 genes that were differentially regulated by butyrate using a combined cutoff of ΔΨ >0.20 (i.e., the posterior distribution over the change in Psi for each event) and Bayes factor >10. The Bayes factor represents the weight of the evidence in the data in favor of differential expression. For example, Bayes factor =2 means that the isoform is two times more likely to be differentially expressed. Of these events, 50 isoforms from 36 genes were supported by at least 3 pair-wise comparisons (Table 4); 29 of the 36 genes had two mRNA isoforms while the rest had at least 3 isoforms. The mRNA-level expression of defensin, beta 1 gene (DEFB1) was not detected in control cells but significantly up-regulated by butyrate (FDR <0.001). This gene had two isoforms. Isoform #1 had 3 exons and was not detectable in control cells but barely detectable in butyrate-treated cells. However, Isoform#2 had 2 exons and was strongly induced by butyrate (Fig. 1). Similarly, IL-18 (ENSBTAG00000000277) had 2 isoforms with the same number of exons. These 2 isoforms differed in the exon structure, nevertheless. Isoform#1 had a similar number of assigned sequence reads while Isoform#2 had approximately 10 times more assigned reads in butyrate-treated cells (252.08±30.51, mean ± sd) than in control cells (25.94±13.69) (P<0.0001, Fig. 2). Isoform #1 of origin recognition complex, subunit 1 (ORC1, transcript# ENSBTAT00000052505) had mean read counts of 168.26±52.75 (± sd) and 1239.75±48.02 in butyrate and control groups, respectively, and was strongly repressed by butyrate. Isoform#2 of ORC1 remained unchanged. Histone deacetylase 10 (HDAC10) had 2 isoforms with the read count ratio between dominant (ENSBTAT00000014602) and minor (ENSBTAT00000042583) isoforms greater than 150 in the control cells. Similarly, the dominant isoform was significantly repressed (P<0.00001), while the minor isoform was not affected by butyrate. However, the expression of both isoforms of HDAC5 was strongly enhanced by butyrate treatment (data not shown). For genes with ≥3 isoforms, one or 2 isoforms were often differentially regulated by butyrate. As Figure 3 shows, the expression level of Isoforms# 1 and 3 of aminoacylase 1gene (ACY1) was unchanged but that of Isoform#2 was strongly repressed by butyrate (Fig. 3). On the other hand, Isoform#2 of coiled-coil domain containing 24 (CCDC24) was little changed in response to butyrate treatment while Isoforms#1 and 3 were significantly induced by butyrate (Fig. 4). Moreover, differential expression of 6 selected transcript isoforms induced by butyrate, including transcripts such as ENSBTAT00000009005 (LGALS9), ENSBTAT00000010687 (CLSTN3), ENSBTAT00000012035 (MX1), ENSBTAT00000020401 (NASP), ENSBTAT00000036545 (GUK1), and ENSBTAT00000061158 (TACC2), were confirmed using RT-PCR.

Bottom Line: This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing.Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control.Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Center for Research in Biological Systems, University of California San Diego, San Diego, California, United States of America.

ABSTRACT
As a signaling molecule and an inhibitor of histone deacetylases (HDACs), butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control (CT) groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001) at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor) and Exon#11 (Acceptor) in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

Show MeSH