Patterns of Histone Methylation and Chromatin Organization in Grapevine Leaf. Rachel Schwope EPIGEN May 24-27, 2016
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1 Patterns of Histone Methylation and Chromatin Organization in Grapevine Leaf Rachel Schwope EPIGEN May 24-27, 2016
2 What does H3K4 methylation do? Plant of interest: Vitis vinifera Culturally important An economically vital organism for Italy and beyond Scientifically intriguing A clonal organism whose genome has not undergone meiosis or its developmental program in centuries.
3 Chromatin the structural and functional complex of DNA and binding proteins ase.org.uk
4 What does H3K4tomethylation do? states Chromatin is specialized maintain genomic A recent ChIP-Seq meta-study identified 9 states of chromatin in Arabidopsis (Sequeira-Mendes, Plant Cell 2014) 4 types = high transcription 5 types = low transcription Promoter and CDS-rich Intergenic and TE-rich (Sequeira-Mendes, Plant Cell 2014)
5 What does H3K4 methylation do? What defines a Chromatin State? From Figure 1B. 1) DNA methylation (CG, CHG, CHH) 2) Post-translational modification of histones (H3K9ac, etc.) 3) Histone variants (H3.1, H2AZ, etc.) ( (Sequeira-Mendes, Plant Cell 2014)
6 What does H3K4 methylation do? Functions of Specific Histone Modifications at H3 H3K4me3 Recruits transcription initiation factors Promotes open conformation H3K9me2 In Arabidopsis, bound by CMT3 DNA methylase In maize, increased at transposons and some genes H3K27me3 Associated with Polycomb In maize, found in genedense chromatin arms, not at pericentric chromatin Layman and Zuo, Front. Cell. Neurosci., 07 January 2015
7 Chromatin Immunoprecipitation Methodology Chromatin from fixed leaf tissue or nuclei ChIP with Abcam Plant Kit Library production (Nugen Ultralow) Bowtie Alignment MACS2 Peak Call 125 bp PE Illumina Sequencing Quantification and Quality Control
8 Where is H3K4me3 found in grapevine leaves? Peaks are generally at the 5 ends of genes Have a broader shoulder at the 3 side of the peak Chromosome 2: 3,198,150..3,225,396 >>>>>>>>>>>>> <<<<<<<<<<<< >>>>>>>>>>>>> <<<<<<<<<<<<<<<<<< 27 kb
9 Vitis genes sorted according to size 100% of gene length H3K4me3 distribution across genes H3K4me3 distribution across genes H3K4me3 coverage over gene body As gene size increases, H3K4me3 covers a proportionally smaller amount of the gene >>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>
10 Fold Enrichment What does H3K4 methylation do? How Enriched is H3K4me3 in Grapevine leaf? ,00 Enrichment of H3K4me3 peaks in grapevine leaf 30,00 25,00 20,00 15,00 10,00 5,00 0,00 Total Peaks
11 What does H3K4 methylation do? H3K4me3 Enrichment generally increases with Peak Length
12 What But does only H3K4 correlates methylation weakly with do? Transcript Accumulation FPKM data from M. Miculan
13 Peak What length does also H3K4 correlates methylation weakly do? with Transcript Accumulation FPKM data from M. Miculan
14 Where else do we find H3K4me3 enrichment? Where else do we find H3K4e3? Long non-coding RNAs: at least 200 bp long 2044 intergenic long-ncrnas in Vitis genome (Genoscope) In leaf, 219 of these (11%) correlate with H3K4me3 signal lncrna Genes <<<<<<<<<<<< <<<<<<<<
15 What does H3K4 methylation do? What are the lncrna species co-occur with H3K4me3? Total: 219 lncrnas 15 of these encode mirnas (mirbase.org) 204 have no discernable mirna structure
16 What does H3K4 methylation do? How do Heterochromatic modifications compare?
17 What does H3K4 methylation do? Where do we find H3K9me2 and H3K27me3? K9me2 51.8% 26.0% 3.3% Total peaks = 11,487 LTR-Gypsy LTR-Copia RIL K27me3 50.0% 26.5% 2.4% Total peaks = 12,270
18 What does H3K4 methylation do? Many, but Not All Highly Repetitive TEs are Enriched kmers H3K27me3 H3K9me2 TEs kmers H3K27me3 H3K9me2 TEs
19 What does H3K4 methylation do? Computational Background Test What happens when I ask MACS2 to find peaks from an unenriched alignment? Input 1 Peak Call Input 2
20 What does H3K4 methylation do? Separating Signal from Noise kmers Input mock H3K27me3 H3K9me2 TEs True Positives False Positives
21 What What is the does DNA H3K4 methylation methylation state at do? loci with modified histones? Genome-wide average methylation H3K4me3 H3K9me2 CG CHG CHH CG CHG CHH CG CHG CHH CG CHG CHH M. Celii
22 Vitis results What support does H3K4 Arabidopsis methylation data that do? DNA and histone methylation form feedback loop Adapted from Stroud et al., 2013
23 What does H3K4 methylation do? CHH CHG CG Chr 4 H3K9me2 H3K27me3 H3K4me3 Chr 3 Transposon Elements Genes Chr 2 M. Celii Chr 1
24 Looking What does the Global H3K4 Chromatin methylation Environment do? with Hi-C Hi-C: a method of inferring the global three-dimensional relationships from paired-end sequence data
25 What does H3K4 methylation do? Hi-C Methodology In the lab: Adapted from Lieberman et al, 2009 In silico: Integrative Genomics and Bioinformatics Core at the Salk Institute
26 A. Tocci Chr 8 What does H3K4 methylation do? Loose Structural Domain Compact Structural Domain Chr 8 Each chromosome shows a unique pattern of organization
27 What does H3K4 methylation do? ChIP data corresponds with 3D structure LSD CSD Chr 8 H3K4me3 H3K9me2 H3K27me3
28 A. Tocci Chr 17 What does H3K4 methylation do? Loose Structural Domain Compact Structural Domain Chr 17 Each chromosome shows a unique pattern of organization
29 What does H3K4 methylation do? ChIP data corresponds with 3D structure LSD CSD Chr 8 Chr 17 H3K4me3 H3K9me2 H3K27me3
30 A. Tocci Chr 4 What does H3K4 methylation do? Loose Structural Domain Compact Structural Domain Chr 4 Each chromosome shows a unique pattern of organization
31 What does H3K4 methylation do? ChIP data corresponds with 3D structure LSD CSD Chr 4 Chr 8 H3K4me3 H3K27me3 H3K9me2
32 What does H3K4 methylation do? Chromatin Organization is Highly Stable Biological Replicate 1 Biological Replicate 2 Chr Chr
33 Pinot Noir Genomic Contact Map Easily visualize the actual locations of unassigned regions of the genome assembly A. Tocci
34 What does H3K4 methylation do? Conclusions and Future Directions 1. Much to uncover with ChIP-Seq H3K4me3 results = high confidence Allele specific analysis in progress Other histone modifications will require finer analyses 2. Hi-C shows chromatin structure very stable Try varieties other than Pinot Noir do they have the same organization? Use Hi-C to improve genome assembly (in progress - Aldo Tocci)
35 What does H3K4 methylation do? Acknowledgements IGA Prof. Michele Morgante Emanuele de Paoli Fabio Marroni Aldo Tocci Mirko Celii Mara Miculan Gabriele Magris IGA Technology Services Emanuela Aleo Federica Magni Giacomo Prete Lab and Administration Giusi Zaina Nicoletta Felice Stefano Grasso Supported by the ERC project NOVABREED - Novel variation in plant breeding and the plant pan-genomes (Grant agreement no.: )
36 What does H3K4 methylation do? CHH CHG CG Chr 4 H3K9me2 H3K27me3 H3K4me3 Chr 3 Transposon Elements Genes Chr 2 Chr 1
37 Average # of peaks per 50 kb window Hi-C shows What H3K4me3 does H3K4 is located methylation preferentially do? in Loose Structural Domains A. Tocci Average # of genes per 50 kb window H3K4me3 Peaks Genes
38 Average # of peaks per 50 kb window A. Tocci What does H3K4 methylation do? H3K9me2 distribution mimics that of total TEs Average # of TEs per 50 kb window H3K9me2 Peaks Transposon Elements Ratio = 1.31 Ratio =
39 What does H3K4 methylation do?
40 20-mer Kmer count Transposons What does with H3K4 K9me2 methylation or K27me3 do? have higher k-mer count K-mer count of TEs from Chromosome 2 TEs with H3K9me2 TEs with H3K27me2 All TEs in Chr2
41 Chromatin Immunoprecipitation Enriching for Antibody-associated DNA with Chromatin Immunoprecipitation
42 What does H3K4 methylation do? H3K9me2 Enrichment generally increases with Peak Size
43 Transposons What does with H3K4 K9me2 methylation or K27me3 do? have higher k-mer count 20-mer K-mer count K-mer count of TEs in Chromosome 2 TEs with H3K27me3 TEs with H3K9me2 All TEs in Chr2
44 How do What heterochromatic does H3K4 modifications methylation at do? histones relate to DNA methylation? DNA transposon
45 What does H3K4 methylation do? How do heterochromatic modifications at histones relate to DNA methylation?
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