Article Review. DNA-methylation effect on co-transcriptional splicing is dependent on GC-architecture of the exon-intron structure

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1 Article Review DNA-methylation effect on co-transcriptional splicing is dependent on GC-architecture of the exon-intron structure 15 March 2013 Genome Research 1

2 Article Genome Research, 15 March 2013, pre-print Publication in 6 months Sahar Gelfman, Noa Cohen, Ahuvi Yearim and Gil Ast Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Israel 2

3 Field of interest Methylation influence on exons recognition and splicing mechanism. 3

4 Quality Pluses: Field overview Lots of references to related works, data sets Minuses: Not well-written Results aren t clear Only average effect was considered Observes correlation but doesn t explain it 4

5 Already known Splicing is co-trascriptional introns removed while transcript is attached to the DNA by RNA pol II GC content Increased in exons mcg/cg level in exons higher than in introns (Schwartz 2009) The level of mcg/cg decreases with increasing GC levels (..) DNA-methylation Affects transcription Strong exon-boundaries marker (Lauren 2010) Possible splicing regulator (Lauren 2010) Depletion of mc in Hox genes --> removing pol II stalling and facilitating transcriptional elongation and efficient splicing (Tao et al. 2010) 5

6 Background Article: Lister et al. (2009) revealed that there is a higher level of CpG-methylation on exons than on flanking introns. However, this difference was dissolved when values were divided by cytosine composition, since exons have a higher GC content, on average, than their flanking introns (Schwartz et al. 2009) Mean mc/c Profiles Over Genomic Regions. Gene body regions were divided in 20 bins from 5' to 3' end, and the mean mc/c level within each bin for each methylation type was determined (mcg/cg black, mchg/chg red, CHH/CHH blue). (Lister et al. (2009)) 6

7 Already known Nucleosome occupancy Increased along exons (Schwartz et al. 2009) Strongly biased towards high GC content (..) Positioning of nucleosomes Partially determined by the DNA sequence and GC content (..) Chromatin remodelers changes positioning (..) DNA-methylation - chromatin remodeler: rigid nucleosomal conformation binding of methyl-binding proteins (MBPs) Nucleosome - roadblock for polii (polii pauses, unwind DNA, release nucleosome, transcription elongation) (..) Transcriptional pausing may allow co-transcriptional recognition of splicing signals in the pre-mrna (de la Mata et al. 2003). 2 models: Intron recognition, Exon recognition (Amit et al. 2012) 7

8 Hypothesis Change in nucleosome occupancy that accompanies DNA methylation might have an indirect influence on pol II elongation rate and co-transcriptional splicing. RNA PolII pauses at splice sites and let other factors (probably methylation binding proteins) to recognize splice site and take decision to do alternative splicing or not. 8

9 Main Question 1. Could the roles of epigenetic modifications in splicing be a side effect of the GC content of the exons? 2. How could one isolate the measured levels of DNA-methylation and nucleosome occupancy on exons, and conclude a biological role unbiased by GC-content? 9

10 Exons groups Exons by GC content architecture 1. Differential GC - significantly higher GC in exons than flanking introns (p-value < 0.05) 2. Leveled GC - same GC level Exons by Splicing behaviour Alternative - optional included in gene mrnas Constitutive - always in included in gene mrnas 10

11 Agenda Investigations Role of mcg as exons bounds marker Role of mcg in exons recognition and inclusion in mrna Influence of CG dinucleotide on chromatine organization (nucleosome positioning) Role of GC content in exons recognition and chromatin organization Article results Article Bottle necks 11

12 mcg As Exons Marker Differential exons exon based splicing recognition 15,874 exons (RefSeq,? by Amit 2012) Leveled exons intron based splicing recognition 16,269 exons (RefSeq,? by Amit 2012) H1 cells, Salk methylome data (Lister 2009) GC content ATTGGGGCAC - 60% GC content, 0% CG ACGCCAATCG - 60% GC content, 40% CG mcg/cg avg. methylation level per base for each group = (sum of methylation level at each pos) / (exons number with CG in this position) [discussion:?] 12

13 mcg As Exons Marker mcg/cg significantly high in exons vs introns, both groups (p-value < 2.2 x ) intron vs. exons mcg/cg methylation level increase: +6% - differential exons +30% - leveled exons!!!! 13

14 mcg As Exons Marker Basic DNA methylation level by base calls ([?:mc/c]) - strong exons signal CG abundance - strong exons signal in agreement with already know CG di-nucleotide abundance works (Karlin 1995, Gentles 2001) 14

15 mcg As Exons Marker RRBS (reduced representation bisulfite sequencing) - most loci and CpG-rich promoters regions; approx. 5% CpG Mouse - RRBS data exons - nearly same results, noise!!! 15

16 mcg As Exons Marker Different tissues: show nearly same pattern 16

17 mcg As Exons Marker Nucleosome occupancy on exons vs flanking introns (Amit et al. 2012): differential exons : 50% increase leveled exons: 10% increase Although nucleosomes occupancy differs both have strong methylation signal on exons. Group weakly marked by nucleosomes is more significantly marked by methylated CpG 17

18 mc For Exon Recognition mcg/cg level in alternatively and constitutively spliced exons examined to evaluate DNA methylation role in recognition Exons splicing types (alt/const) obtained from RNA-Seq (H1 cellline) SpliceTrap tool - For every exon it quantifies the extent to which it is included, skipped or subjected to size variations due to alternative 3 / 5 splice sites or Intron Retention. In addition, SpliceTrap can quantify alternative splicing within a single cellular condition, with no need of a background set of reads. SpliceTrap approaches the expression-level estimation of each exon as an independent Bayesian inference problem exons with canonical splicing differential exons : 7413 (5734 const, 1679 alternative) leveled exons: 6037 (4936 const, 1101 alternative) 18

19 mc For Exon Recognition exon-intron CG structure as expected in constitutive/alternative exons groups 19

20 mc For Exon Recognition mcg/cg general signal pattern same for differential & leveled GC constitutive exons (multiple t-test, p-value < 2.2x10-16 ) mcg/cg lower in alternative vs. constitutive exons (multiple t-test, p-value < 2.2x10-16 ) 20

21 mc For Exon Recognition Nucleosome occupancy signal Differential GC exons - strong Leveled GC exons - weak 21

22 CG & Chromatine Structure High mcg/cg level was observed near 3, 5 splice sites Does presence of CG dinucleotide in splice signal consensus lead to high mcg/cg? Control dataset pseudo exons (Ke et al. 2011): Pseudo exons - intronic sequences having lengths between 50 and 250 nt and splice site motif score close to splice sites scores In addition, pseudo exons had to be at least 100 nt away from the closest real exon Pseudo exons not biased to any GC differential in exons vs introns 22

23 CG & Chromatine Structure Pseudo exons not biased to any GC differential in exons vs introns 23

24 CpG & Chromatine CG abundance higher in lev., diff. but same in pseudo exons CG high increased (mc in 70% of cases) -> 3 : -5; 5 : -2, +4 3 peaks positions may have a regulatory role. 24

25 CpG & Chromatine Peaks role as chromatin remodelers? Was shown that certain DNA sequences with high affinity binding to the histone can direct nucleosome positioning (Lowary and Widom 1998) DNA methylation may have an intrinsic effect on nucleosome positioning on the DNA (Chodavarapu et al. 2010; Cedar and Bergman 2012) No method found for single by methylation & chromatin organization quantification. CD4+ T cells nucleosomes data (Schones et al. 2008).[discussion:] running average, on 20 nt window 25

26 CpG & Chromatine Methylated CG: ES cells % CG methylated (Lister 2009) [JetBioLabs: 74-87%] 70-88% CG in differentiated & leveled GC groups methylated (Fig.1) => CG dinucleotide acts as representative of methylated position. [discussion:!!!] 70-88% is mlevel of CG-not same %CG methylated Exons sub groups based on mc peak positions CG composition group: CG at the peak position GC composition group: C G, but not GC at the peak position not-c composition group: other nucleotides ( i.e AA,AT,AC,AG..) AG composition group (not-c sub-group): AG at peak position GC group - control for CG group (same GC content) AG group for 5 : -2 position 26

27 CpG & Chromatine Leveled exons - increased nucleosome occupancy when Any of the 3 peak positions is a CG in the group Near 3 peak positions for CG compared to others (multiple t-tests, p-value < ) other positions - trend of increased nucleosome occupancy when a CG is present but the effect is smaller that at the peak. 27

28 CpG & Chromatine Differential exons - less nucleosome occupancy Depleted signal when -5 at 3 site is CG comparing with GC (p-value < 0.006) Small increase for -2 (p-value < ) and +4 (p-value < 0.043) at 5 site other positions - increasing occupancy not observed, occupancy is not significantly affected by dinucleotide composition near the splice site. 28

29 CpG & Chromatine Question: Does CG peaks express some special GC-content signal? CG at 3 peak positions doesn t significantly affect GC content of any sub-group Cannot explain strong nucleosome occupancy signal of CG group in leveled exons 29

30 CpG & Chromatine 3 CG peaks detected close to splicing sites CG at peaks are frequently methylated => Probably directs nucleosomes binding mediated by MBP (methylation binding proteins) Nucleosomal signal Differential CG group - weak CG peaks effect Leveled CG group - strong CG peaks nucleosomal signal => Exons recognition mechanism may depend on the GC environment of exon/gene/location (will be in further works) 30

31 GC Content Role Perhaps difference in epigentic pattern is chromosomes specific due to different GC content? Are Exon-intron patterns (Fig 1,2) dependent on GC content? Isochore maps (Costantini et al. 2006) Isochore maps - regions >= 300 K bases with homogeneous GC content. Core (GC > 46%) and Desert (GC < 46%) parts 31

32 GC Content Role Leveled & Differential group not equally distributed along genome Differential - more in lower GC chromosomes Leveled - in high GC chromosomes 32

33 GC Content Role Exons H1 were divided in core (53 102) and desert (85 979) exons exons RNA-seq data contains only => alt. and const. exons were considered as EST-based (expressed sequence tag) 33

34 GC Content Role Methylation is strong exons marker for both core and desert exons 34

35 GC Content Role Methylated CG level drops (desert & core groups) in alternative exons vs. const. exons Mean mcg level increase for const. vs. alt exons groups core exons +24% mcg level (6 443 of ) desert exons +19% mcg level (9 649 of alt.) 35

36 GC Content Role Nucleosomes occupancy low GC regions: often on exons than introns high GC regions: spread along both exons and introns 36

37 Results Alternative Constitutive Differential Leveled GC content Nucleosomes occupancy n/a n/a high exons marking weak exons marking but CG-related signal presence at 3 positions GC-content dependent Methylation Lower mcpg level on alternative comparing to const. spliced exons Clear marking; Stronger signal on leveled exons; Intronic mc reduction near splice sites. Exons marking GC content independent 37

38 Results GC content bias elimination - Leveled & Diff exons Group weakly marked by nucleosomes is more significantly marked by methylated CpG In a Leveled GC architecture methylated CpGs accompanied higher inclusion of exons, i.e. exons are recognized and aren t removed by splicing. Not the absolute methylation value that distinguishes const. vs alt. exons, but the differential in the ratio of mcpg/cpg between exon and introns, which is also dependent in the general GC environment Supports for the role of DNA methylation as a splicing regulator 38

39 Discussion =?=> CpG methylation can be a part of the code that allows the splicing machinery to locate exons that have no GC differential. Identified a strong decrease in methylated CpGs in alternative exons and their flanking sequences compared with constitutive exons =?=> lower DNA methylation levels of the whole intron-exon-intron strip are associated with suboptimal recognition of alternatively spliced exons Effect on splicing of a strong DNA methylation signal in a leveled GCarchitecture may be indirectly through pol II stalling ( =?=> detected peak in CG abundance) =?=> peaks in CpG methylation at the 5 and 3 splice sites act as the central area binding for the nucleosomal splicing barrier, while the drop in methylated CpGs in intronic flanking regions of leveled GC exons point to the entry/exit regions of the nucleosome. 39

40 Article bottle necks Only CG methylation in ES cells considered, much reasonable to check CG methylation in differentiated cells mcg/cg average methylation level is a vague characteristics. It shows methylation variability at position among different cells in passage. More over Lister (2009) showed that there are large PMM domains in DNA. Seems mcg/cg signal in all cases was induced by higher CG density and signal Nucleosomes binding isn t clear, Chip-Seq based methods provides positions with precision about +/ bp Authors replace mcg with just CG presence in exons. Such trick seemed to be incorrect. Hard to interpret trends in average for a particular exon. 40

41 Tel-Aviv University Differential & Leveled GC content, short/long introns. 2 splicing models (intron/exon recognition). (Amit et al. 2012) Chromatin organization role in splicing (Schwartz et al. 2009) 41