Details
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Type: Task
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Status: Closed (View Workflow)
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Priority: Major
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Resolution: Done
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Affects Version/s: None
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Fix Version/s: None
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Labels:None
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Story Points:3
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Sprint:Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11, Spring 5 2022 Feb 28 - Mar 11
Description
To understand the significance of our results, we need to learn the current state of knowledge in the field.
For this task, assemble and summarize original research articles published in the last two to three years that investigated how methylation affects alternative splicing.
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Nowlan Freese
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Nowlan Freese
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Field | Original Value | New Value |
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Epic Link | IGBF-3039 [ 21553 ] |
Nowlan Freese
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Status | To-Do [ 10305 ] | In Progress [ 3 ] |
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Status | In Progress [ 3 ] | To-Do [ 10305 ] |
Nowlan Freese
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Status | To-Do [ 10305 ] | In Progress [ 3 ] |
Ann Loraine
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Sprint | Spring 2 2022 Jan 18 - Jan 28 [ 137 ] | Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11 [ 137, 138 ] |
Ann Loraine
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Rank | Ranked higher |
Nowlan Freese
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Nowlan Freese
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Story Points | 2 | 3 |
Nowlan Freese
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Attachment | 3069.enl [ 17091 ] |
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Status | In Progress [ 3 ] | Needs 1st Level Review [ 10005 ] |
Nowlan Freese
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Assignee | Nowlan Freese [ nfreese ] |
Ann Loraine
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Sprint | Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11 [ 137, 138 ] | Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11, Spring 4 2022 Feb 14 - Feb 25 [ 137, 138, 139 ] |
Ann Loraine
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Rank | Ranked higher |
Ann Loraine
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Sprint | Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11, Spring 4 2022 Feb 14 - Feb 25 [ 137, 138, 139 ] | Spring 2 2022 Jan 18 - Jan 28, Spring 3 2022 Jan 31 - Feb 11, Spring 5 2022 Feb 28 - Mar 11 [ 137, 138, 140 ] |
Ann Loraine
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Status | Needs 1st Level Review [ 10005 ] | First Level Review in Progress [ 10301 ] |
Ann Loraine
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Assignee | Ann Loraine [ aloraine ] |
Ann Loraine
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Status | First Level Review in Progress [ 10301 ] | Ready for Pull Request [ 10304 ] |
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Status | Ready for Pull Request [ 10304 ] | Pull Request Submitted [ 10101 ] |
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Status | Pull Request Submitted [ 10101 ] | Reviewing Pull Request [ 10303 ] |
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Status | Reviewing Pull Request [ 10303 ] | Merged Needs Testing [ 10002 ] |
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Status | Merged Needs Testing [ 10002 ] | Post-merge Testing In Progress [ 10003 ] |
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Resolution | Done [ 10000 ] | |
Status | Post-merge Testing In Progress [ 10003 ] | Closed [ 6 ] |
Ann Loraine
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Assignee | Ann Loraine [ aloraine ] | Nowlan Freese [ nfreese ] |
Epigenetic differences in an identical genetic background modulate alternative splicing in A. thaliana
How stable and temperature-dependent variations in DNA methylation and nucleosome occupancy influence alternative splicing (AS) remains poorly understood in plants. To answer this, we generated transcriptome, whole-genome bisulfite, and MNase sequencing data for an epigenetic Recombinant Inbred Line (epiRIL) of A. thaliana at normal and cold temperature. Our transcriptome data revealed that differential DNA methylation and nucleosome occupancy modulate expression levels of many genes and AS in response to cold. Collectively, DNA methylation and nucleosome levels exhibit characteristic patterns around intron-exon boundaries at normal and cold conditions, and any perturbation in them, in an identical genetic background is sufficient to modulate AS in Arabidopsis.
Furthermore, the AS analysis showed that epigenetic differences between Col-0 and epiRIL-368 induced fewer but contrasting changes under similar temperature conditions (22 C and 4 C) (one-way ANOVA p-value = 1.7492e-09 for DAS genes). For instance, the number of identified DAS genes between Col-0 versus epiRIL-368 was 305 and 311 at 22 C and 4 C, respectively (Fig. 1A a,b).
Intrestingly, there is no overlap between DEGs and DAS genes between Col-0 and epiRIL-368 at 22 C (hypergeometric test p-value =0.198) and 4 C (hypergeometric test p-value = 2.520e-05) (Fig. 1B a-b). Whereas, this number significantly increases (Col-0 at 22 C versus epiRIL-368 at 4 C; 7.1%; hypergeometric test p-value 0.011) when epigenetic variations and cold stress adds on together (Fig. 1B c).
Further, we performed gene functional enrichment analysis for DEGs and DAS genes (Supplementary File S1) for all three gene ontology (GO) terms i.e. Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) (Fig. S2). Among DEGs, the most significant (FDR <0.05) terms involved transcription regulation, Pol II processivity, cold and other such as response to abscisic acids, are highly enriched (Fig. S2 A) in different contrasting groups.
Therefore, we reasoned those variations at the methylation and nucleosome (epigenetic) levels may affect AS because of epigenetic differences between Col-0 and epiRIL-368 ecotypes under different temperatures. To further investigate these variations, we performed WGBS for epiRIL-368 plants grown at 22 C and 4 C. We identified high confidence differentially methylated regions (hc_DMRs) (Fisher's exact test, p-value <0.01) in comparison to 54 Columbia (Col) lines of Arabidopsis using the hc_DMR caller pipeline developed by the Jacobsen group at the University of California [60].
The high number of hypomethylated regions suggests relatively lower methylation levels in epiRIL-368 compared to Col ecotype (Fig. 2A). Among all hcDMRs, 22,968 were hypomethylated regions in the epiRIL-368 line as compared to Col lines (Fig. 2A).
We first calculated AS event inclusion levels (PSI) for a total of 43,953 AS events identified in the reference annotation file of Arabidopsis, followed by the differences in their inclusion (PSI) among different contrast groups. Differential AS events analysis suggests that epiRIL-368 display significant (p-value <0.05) differences in 474 and 516 AS events compared to Col-0 at 22 C and 4 C, respectively (Fig. 2C; Supplementary File S4).
Different types of AS events detected in our analysis show an overall similar distribution observed previously in Arabidopsis [9,11] where intron retention (IR) events are the most prevalent, followed by usage of the alternative acceptor (A3'SS) and alternative donor (A5'SS) sites, and exon skipping (ES) (Fig. 2C; Supplementary File S4).
The CpG, CHG, and CHH methylation levels generated using methylpy [65] were plotted around the donor sites (exon-intron; 5'SS), the acceptor sites (intron-exon; 3'SS), and the exons for epiRIL-368 at 22 C and 4 C (Fig. 3A; Fig. S3). We observed a sharp drop in CpG methylation at both splice sites (5'SS and 3'SS; Fig. 3A a-b) suggesting its role in transcription and splicing dynamics by affecting Pol II processing around 5'SS and 3'SS as compared to flanking regions. DNA methylation around exons always shows a higher methylation level and can be differentiated from their flanking regions (introns, especially splice sites) (Fig. 3A; Fig. S3A). Regardless of temperature treatment, we also found the level of methylated CpG dinucleotides (mCpG) is higher in exons as compared to flanking regions including introns and splice sites (Fig. 3A).
Next, we looked at the function of the genes with high confidence differentially methylated regions (hcDMRs), differential nucleosome positioning (DNPs), and differential splice junctions (DSJs) in our dataset. We first identified the genes with hcDMRs, and DNPs in addition to genes with DSJs for the contrasting groups Col-0 at 22 C versus epiRIL-368 at 22 C, and Col-0 at 22 C versus epiRIL-368 at 4 C. We divided genes into three groups including genes with hcDMRs (hcDMR genes), genes with significant DNPs (DNP genes) and genes with significant DSJs (DSJ genes). Finally, significantly overlapping (Fig. 4A) genes between hcDMR, DNP, and DSJ genes for the contrasting groups Col-0 at 22 C versus epiRIL-368 at 22 C, and Col-0 at 22 C versus epiRIL-368 at 4 C were selected for gene ontology (GO) functional enrichment analysis.