presentazione standard di powerpoint...2018/07/10  · long non-coding rnas: at least 200 bp long...

Rachel Schwope

EPIGENMay 24-27, 2016

Patterns of Histone Methylation and Chromatin Organization in Grapevine Leaf

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.

Chromatin – the structural and functional complex of DNA and binding proteins

ase.org.uk http://www.structuralbiology.uzh.ch/

What does H3K4 methylation do?Chromatin is specialized to maintain genomic states

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

(Sequeira-Mendes, Plant Cell 2014)

Intergenic andTE-rich

Promoter andCDS-rich

What does H3K4 methylation do?What defines a Chromatin State?

(Sequeira-Mendes, Plant Cell 2014)

1) DNA methylation (CG, CHG, CHH)2) Post-translational modification of histones (H3K9ac, etc.)3) Histone variants (H3.1, H2AZ, etc.)

(www.promega.com)

From Figure 1B.

What does H3K4 methylation do?

H3K4me3

Functions of Specific Histone Modifications at H3

H3K9me2 H3K27me3

• Recruits transcription initiation factors

• Promotes openconformation

• In Arabidopsis, bound by CMT3 DNA methylase

• In maize, increased at transposons and some genes

• Associated with Polycomb

• In maize, found in gene-dense chromatin arms, not at pericentric chromatin

Layman and Zuo, Front. Cell. Neurosci., 07 January 2015

Chromatin Immunoprecipitation Methodology

Chromatin from fixed leaf tissue

or nuclei

ChIP with AbcamPlant Kit

Library production

(Nugen Ultralow)

Quantification and Quality

Control

125 bp PE Illumina

Sequencing

Bowtie Alignment

MACS2 Peak Call

Peaks are generally at the 5’ ends of genes

Have a broader “shoulder” at the 3’ side of the peak

Where is H3K4me3 found in grapevine leaves?

Chromosome 2: 3,198,150..3,225,396

27 kb

>>>>>>>>>>>>> <<<<<<<<<<<< >>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<

H3K4me3 distribution across genes

As gene size increases, H3K4me3 covers a proportionally smaller amount of the gene

H3K4me3 distribution across genes

100% of gene length

Vit

isge

nes

so

rted

acc

ord

ing

to s

ize

H3K4me3 coverage over gene body

>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>

What does H3K4 methylation do?How Enriched is H3K4me3 in Grapevine leaf?

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

1

81

4

16

27

24

40

32

53

40

66

48

79

56

92

65

05

73

18

81

31

89

44

97

57

10

57

0

11

38

3

12

19

6

13

00

9

13

82

2

14

63

5

15

44

8

16

26

1

17

07

4

17

88

7

18

70

0

19

51

3

20

32

6

Fold

Enri

chm

en

t

Total Peaks

Enrichment of H3K4me3 peaks in grapevine leaf

What does H3K4 methylation do?H3K4me3 Enrichment generally increases with Peak Length

What does H3K4 methylation do?…But only correlates weakly with Transcript Accumulation

FPKM data from M. Miculan

What does H3K4 methylation do?Peak length also correlates weakly with Transcript Accumulation

FPKM data from M. Miculan

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

Where else do we find H3K4me3 enrichment?

<<<<<<<<<<<< <<<<<<<<Genes

lncRNA

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

What does H3K4 methylation do?How do Heterochromatic modifications compare?

What does H3K4 methylation do?Where do we find H3K9me2 and H3K27me3?

LTR-Gypsy LTR-Copia RIL

K9me2

K27me3Total peaks = 12,270

Total peaks = 11,48751.8%

50.0%

26.0%

26.5%

3.3%

2.4%

What does H3K4 methylation do?Many, but Not All Highly Repetitive TEs are Enriched

kmers

H3K27me3

H3K9me2

TEs

kmers

H3K27me3

H3K9me2

TEs

What does H3K4 methylation do?Computational Background Test

What happens when I ask MACS2 to find peaksfrom an unenriched alignment?

Input 1

Input 2

Peak Call

What does H3K4 methylation do?Separating Signal from Noise

kmers

H3K27me3

H3K9me2

TEs

‘Input mock’

‘True Positives’ ‘False Positives’

What does H3K4 methylation do?What is the DNA methylation state at loci with modified histones?

H3K4me3 H3K9me2

CG CHG CHH CG CHG CHH CG CHG CHH CG CHG CHH

Genome-wide averagemethylation

M. Celii

What does H3K4 methylation do?Vitis results support Arabidopsis data that DNA and histone methylation form feedback loop

Adapted from Stroud et al., 2013

What does H3K4 methylation do?

Chr 4

Chr 3

Chr 2

Chr 1

CHH

CHG

CG

H3K9me2

H3K27me3

H3K4me3

Transposon Elements

Genes

M. Celii

What does H3K4 methylation do?Looking at the Global Chromatin Environmentwith Hi-C

http://medcell.med.yale.edu

Hi-C: a method of inferring the global three-dimensional relationships from

paired-end sequence data

http://mol-biol4masters.masters.grkraj.org

What does H3K4 methylation do?Hi-C Methodology

Adapted from Lieberman et al, 2009

In the lab:

In silico:

Integrative Genomics and Bioinformatics Core at the Salk Institute

What does H3K4 methylation do?

Chr 8

Ch

r8

• Each chromosome shows a unique pattern of organization

A. Tocci

“Loose Structural Domain”

“Compact Structural Domain”

What does H3K4 methylation do?

Chr 8

LSD

CSD

ChIP data corresponds with 3D structure

H3K27me3

H3K9me2

H3K4me3

What does H3K4 methylation do?

Chr 17

Ch

r1

7

• Each chromosome shows a unique pattern of organization

A. Tocci

“Loose Structural Domain”

“Compact Structural Domain”

What does H3K4 methylation do?

Chr 8

LSD

CSD

ChIP data corresponds with 3D structure

H3K27me3

H3K9me2

H3K4me3

Chr 17

What does H3K4 methylation do?

Chr 4

Ch

r4

• Each chromosome shows a unique pattern of organization

A. Tocci

“Loose Structural Domain”

“Compact Structural Domain”

What does H3K4 methylation do?

Chr 8

LSD

CSD

ChIP data corresponds with 3D structure

H3K27me3

H3K9me2

H3K4me3

Chr 4

What does H3K4 methylation do?Chromatin Organization is Highly Stable

Biological Replicate 1 Biological Replicate 2

Chr1 2 3 4 5 6 7 8 9 10 Chr1 2 3 4 5 6 7 8 9 10

Pinot Noir Genomic Contact Map

Easily visualize the actual locations of unassigned regions of the genome assembly

A. Tocci

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)

What does H3K4 methylation do?Acknowledgements

IGAProf. Michele MorganteEmanuele de PaoliFabio MarroniAldo TocciMirko CeliiMara MiculanGabriele Magris

IGA Technology ServicesEmanuela AleoFederica MagniGiacomo Prete

Lab and AdministrationGiusi ZainaNicoletta Felice

Stefano GrassoSupported by the ERC project NOVABREED - Novel variation in plant breeding and the plant pan-genomes (Grant agreement no.: 294780)

What does H3K4 methylation do?

Chr 1

CHH

CHG

CG

H3K9me2

H3K27me3

H3K4me3

Transposon Elements

Genes

Chr 2

Chr 3

Chr 4

What does H3K4 methylation do?Hi-C shows H3K4me3 is located preferentially in Loose Structural Domains

2.54

12636

4.89

22098

Ave

rage

# o

f p

eak

s p

er

50

kb

win

do

w

Ave

rage

# o

f ge

nes

pe

r 5

0 k

b w

ind

ow

H3K4me3 Peaks Genes

.709

3524

2.65

11988

A. Tocci

What does H3K4 methylation do?H3K9me2 distribution mimics that of total TEsA

vera

ge #

of

pe

aks

pe

r 5

0 k

b w

ind

ow

H3K9me2 Peaks Transposon Elements

Ave

rage

# o

f TE

s p

er 5

0 k

b w

ind

ow

18.79

12.8

1.51

6819

2.03

10068

Ratio = 1.31 Ratio = 1.46

A. Tocci

What does H3K4 methylation do?

What does H3K4 methylation do?Transposons with K9me2 or K27me3 have higher k-mer count

20

-mer

Km

erco

un

t

K-mer count of TEs from Chromosome 2

TEs with H3K9me2 TEs with H3K27me2 All TEs in Chr2

http://www.biosyn.com/

Chromatin Immunoprecipitation Enriching for Antibody-associated DNA with Chromatin Immunoprecipitation

What does H3K4 methylation do?H3K9me2 Enrichment generally increases with Peak Size

What does H3K4 methylation do?Transposons with K9me2 or K27me3 have higher k-mer count

20

-mer

K-m

erco

un

tK-mer count of TEs in Chromosome 2

TEs with H3K27me3 TEs with H3K9me2 All TEs in Chr2

What does H3K4 methylation do?How do heterochromatic modifications at histonesrelate to DNA methylation?

DNA transposon

What does H3K4 methylation do?How do heterochromatic modifications at histonesrelate to DNA methylation?