Monica C. Sleumer (苏漠)
2012-09-19
Human Genome
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3,101,804,739 base pairs
22 chromosomes plus X and Y
21,224 protein-coding genes
15,952 ncRNA genes
3–8% of bases are under selection
– From comparative genomic studies
• Question: What is the genome doing?
Objectives
• Find all functional elements
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Bound by specific proteins
Transcribed
Histone modifications
DNA methylation
• Use this information to annotate functional regions
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Genes (coding and non-coding)
Promoters
Enhancers
Specific transcription factor binding sites
Silencers
Insulators
Chromatin states
• Cross-reference data from other studies
– Comparative genomics
– 1000 Genomes Project
– Genome-wide association studies (GWAS)
ENCODE projects
• ENCODE pilot project: 1% of the genome 2003-2007
• modENCODE: Drosophila and C. elegans
• ENCODE main project 2007-2012
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1649 dataset-generating experiments
147 cell types
235 antibodies and assay protocols
450 authors
32 institutes
• 31 publications 2012-09-06
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6 in Nature
18 in Genome Research
6 in Genome Biology
1 in BMC Genetics
www.nature.com/encode/category/research-papers
Materials
• 147 types of human cell lines, 3 priority levels
• Tier 1 cell lines: top priority for all experiments
Name
Description
Lineage
Tissue
Karyotype
GM12878
B-lymphocyte, lymphoblastoid,
Epstein-Barr Virus,
mesoderm
1000 Genomes Project
blood
normal
H1-hESC
embryonic stem cells
inner cell
mass
embryonic stem cell
normal
K562
leukemia, 53-year-old female
with chronic myelogenous
leukemia
mesoderm
blood
cancer
• Tier 2 cell lines to be done after Tier 1 (next slide)
• Tier 3: any other cell lines
Tier 2 Cell Lines
Name
Description
Lineage
lung carcinoma epithelium, 58-yearendoderm
old caucasian male
donor B cells: RO01778 and
CD20+
mesoderm
RO01794
CD20+_RO01778 B cells, caucasian
mesoderm
CD20+_RO01794 B cells, African American
mesoderm
neurons derived from H1
H1-neurons
ectoderm
embryonic stem cells
HeLa-S3
cervical carcinoma
ectoderm
HepG2
hepatocellular carcinoma
endoderm
HUVEC
umbilical vein endothelial cells
mesoderm
IMR90
fetal lung fibroblasts
endoderm
skeletal myoblasts from pectoralis
LHCN-M2
mesoderm
major muscle, 41 year old caucasian
MCF-7
mammary gland, adenocarcinoma ectoderm
MonocytesMonocytes-CD14+, leukapheresis
mesoderm
CD14+
from RO 01746 and RO 01826
SK-N-SH
neuroblastoma, 4 year old
ectoderm
A549
Tissue
Karyotype
epithelium
cancer
blood
normal
blood
blood
normal
normal
neurons
normal
cervix
liver
blood vessel
lung
skeletal muscle
myoblast
breast
cancer
cancer
normal
normal
monocytes
normal
brain
cancer
http://encodeproject.org/ENCODE/cellTypes.html
cancer
Methods
RNA-Seq
Different fractions of RNA -> sequencing
CAGE
5’ Capped RNA sequencing
RNA-PET
Sequencing 5’ Cap plus poly-A tail
ChIP-seq
Chromatin immunoprecipitation of a DNA binding protein -> sequencing
DNase-seq
Cut exposed DNA with DNase I -> sequencing
FAIRE-seq
Nucleosome-depleted DNA -> sequencing
RRBS
Bisulphite treatment: unmethylated C->U -> sequencing
3C,5C, ChIA-PET
Chromatin interactions -> sequencing
Results: RNA Sequencing
• 62% of the genome is transcribed into sequences
>200 bp long
– 5.5% of this is exon
– 31% is intergenic – no annotated gene
– Remaining: intronic
• CAGE-seq: 62,403 TSS
– 44% within 100bp of the 5’ end of a GENCODE gene
– Others: exons and 3’ UTRs, significance unknown
• Lots of short ncRNAs: tRNA, miRNA, snRNA etc.
• Further description: Wu Dingming, 9:30
Results: Transcribed and protein-coding regions
• GENCODE reference gene set
– 20,687 Protein-coding
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6.3 alternatively spliced transcripts on average
3.9 protein isoforms on average
Protein-coding exons: 1.22% of the genome
Still more to come: unidentified peptides in mass-spec
– 18,441 ncRNA genes
• 8801 short ncRNA
• 9640 long nc RNA
– 11,224 pseudogenes
• 863 transcribed
ChIP-Seq
Acronym
Description
ChromRem
ATP-dependent chromatin
complexes
DNA repair
DNARep
HISase
Other
Pol2
Factors
analysed
5
3
Histone acetylation,
8
deacetylation or methylation
complexes
Cyclin kinase associated with
1
transcription
Pol II subunit
1 (2 forms)
Pol3
Pol III-associated
6
TFNS
General Pol II-associated
factor, not site-specific
Pol II transcription factor with
sequence-specific DNA binding
8
TFSS
Total
87
119
www.illumina.com/technology/chip_seq_assay.ilmn
ChIP-Seq: Histone modifications
Histone modification or
Signal
variant
characteristics
H2A.Z
Peak
Association
dynamic chromatin
H3K4me1
Peak/region
H3K4me2
Peak
enhancers and other distal elements, also downstream of
transcription starts
promoters and enhancers
H3K4me3
Peak
promoters/transcription starts
H3K9ac
Peak
promoters
H3K9me1
Region
5′ end of genes
H3K9me3
Peak/region
H3K27ac
Peak
Gene repression, constitutive heterochromatin and
repetitive elements
Gene expression, active enhancers and promoters
H3K27me3
Region
H3K36me3
Region
H3K79me2
Region
polycomb complex, repressive domains and silent
developmental genes
Elongation, transcribed portions of genes, 3′ regions after
intron 1
Transcription, 5′ end of genes
H4K20me1
Region
5′ end of genes
Results: ChIP-Seq
• 636,336 binding regions
• 8.1% of the genome
• Sequence-specific TF ChIP-seq:
– 86% of the DNA segments occupied by sequencespecific transcription factors contained a strong
DNA-binding motif
– 55% cases contained the expected motif
• Further description: Qin Zhiyi & Ma Xiaopeng,
13:30
DNase I hypersensitivity
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2,890,000 unique hypersensitive sites (DHSs)
4,800,000 sites across 25 cell types
Tier 1 and tier 2 cell types: 205,109 DHSs per cell type
98.5% of ChIP-seq TFBS within DHSs
Further description: Guo Weilong 12:30, He Chao 14:30
https://www.nationaldiagnostics.com/electrophoresis/article/dnase-i-footprinting
FAIRE-seq
• Like the opposite of ChIP-seq
• Cross-link the nucleosomes to the DNA
– But not the sequence-specific TFs
• Shear the DNA into small pieces
• Remove the protein-bound DNA
• Sequence the non-bound DNA
Gaulton KJ et al, Nature Genetics 42, 255–259 (2010) doi:10.1038/ng.530
DNA methylation
• CpG methylation: regulates gene
expression
– In promoters: gene repression
– In genes: gene transcription
• 1,200,000 methylated CpGs in 82 cell lines
and tissues
– 96% differentially methylated, especially
those in genes
• Unmethylated genic CpG islands associated
with P300 binding , an enhancer-related
histone acetyltransferase
• Allele-specific methylation: genomic
imprinting
• Aberrant methylation in cancer cell lines
• Reproducible methylation outside CpG
dinucleotides
http://www.diagenode.com/en/applications/bisulfite-conversion.php
Chromosome conformation capture
Montavon and Duboule, Trends in Cell Biology (2012) 22:7, 347–354
Results: Chromosome interactions
• Chromosome conformation capture (3C) :
– 5C: 3C-carbon copy
– ChIA-PET
• Identified 127,417 promoter-centred chromatin
interactions using ChIA-PET
– 98% intra-chromosomal
• 2,324 promoters involved in ‘single-gene’ enhancer–
promoter interactions
• 19,813 promoters were involved in ‘multi-gene’
interaction complexes spanning up to several
megabases
• 50–60% of long-range interactions occurred in only one
of the four cell lines
• Further discussion: Li Yanjian, 10:40
Primary Findings
• 80.4% of the human genome is doing at least one of the
following:
– Bound by a transcription factor
– Transcribed
– Modified histone
• 99% is within 1.7 kb of at least one of the biochemical events
• 95% within 8 kb of a DNA–protein interaction or DNase I
footprint
• 7 chromatin states:
– 399,124 enhancer-like regions
– 70,292 promoter-like regions
• Correlation between transcription, chromatin marks, and TF
binding
• Functional regions contain lots of SNPs
– Disease-associated SNPs in non-coding regions tend to be in
functional elements
End of Introduction
Summary of ENCODE elements
• 80.4% of the human genome is covered by at least one
ENCODE-identified element
• 62% of the genome is transcribed
• 56% of the genome associated with histone modifications
• Excluding RNA elements and broad histone elements,
44.2% of the genome is covered
– open chromatin (15.2%)
– transcription factor binding (8.1%)
– 19.4% DHS or transcription factor ChIP-seq peaks across all cell
lines
• 8.5% of bases are covered by either a transcription-factorbinding-site motif (4.6%) or a DHS footprint (5.7%)
– 4.5x the amount of protein-coding exons (1.2%)
– 2x the amount of conserved sequence between mammals
• Estimate: 50% of DHS remain to be found
– Based on saturation curves
Diversity
Diversity vs Conservation: Interactive Figure
Conservation
A high-resolution map of human
evolutionary constraint using 29 mammals
Nature 478, 476–482 (2011)
Diversity
Conservation in Bound Motifs vs Unbound Motifs
Conservation
http://www.nature.com/encode/interactive-figures/nature11247_F1
Model of gene expression – histone marks
Model of gene expression – TF binding
Transcription factor co-associations
Seven major classes of genome states
CTCF
CTCF-enriched
element
CTCF signal , no histone modifications, open chromatin, may
have insulator function, enriched for cohesin components
RAD21 and SMC3
E
Predicted enhancer
Open chromatin, H3K4me1, other enhancer-associated marks,
enriched for EP300, FOS, FOSL1, GATA2, HDAC8, JUNB, JUND,
NFE2, SMARCA4, SMARCB1, SIRT6 and TAL1 sites, nuclear and
whole-cell RNA poly(A) signal
PF
Predicted promoter
flanking
Regions that generally surround TSS segments
R
Predicted repressed
H3K27me3 polycomb-enriched regions, REST, BRF2, CEBPB,
MAFK, TRIM28, ZNF274 and SETDB1 sites or no signal at all
TSS
Predicted promoter
including TSS
H3K4me3, open chromatin, Pol II, Pol III, short RNAs, close to
TSS sites
T
Predicted
transcribed
H3K36me3 transcriptional elongation signal., overlap with gene
bodies, phosphorylated Pol II , cytoplasmic poly(A)+ RNA
WE
Weak enhancer
Similar to the E state, but weaker signals and weaker
enrichments.
Data integration and genome segmentation
Enhancer
Transcribed
Repressed
TSS
RNA expression
Transcription factors
Association between genome states and annotations
Genome segment
Genome segment
Enhancer validation in mouse and fish
Enhancer from K562 cell (leukemia) drives basal promoter with reporter gene in
embryonic mouse blood cells
and medaka fish
Genome segment clustering
6 cell types
Genome cluster function
Genome state
is related to
gene function
Allele-specific expression
Pol II
Txn
Rpn
Correlation of allele-specific signal
by gene
by genomic
segment
Genome-wide association studies
Annotated diseasecausing SNPs
Control SNPs
Selected TFBS tracks
Significant
overlap
Diseases
No genes, but
several TFBS
near the
disease-causing
SNPs
Conclusions
• 80% of human genome annotated with at least
one association
– Protein-binding
– Histone modification
– Transcription
• ENCODE data combination
– Model gene expression
– Genome segmented into 7 types
• Different in each cell line
• ENCODE data combined with other data
– 1000 genomes: see influence of parental DNA
– Genome-wide association studies
Discussion
• 147 types of cells, and the human body has a few thousand
• 80% functional : controversial
– 80% of the genome is being transcribed and/or has a protein bound to
it some of the time
– Heterochromatin: tightly packed repeat sequences
– most of that activity isn’t particularly specific or interesting and may
not have impact
– Important not to overstate the findings
– Ewan Birney: “cumulative occupation of 8% of the genome by TFs”
• Reproducibility
– In exactly the same cell lines, same conditions, different time or place
– Same cell lines, different conditions
– Same cell type, different people
• Cell lines vs tissue
• Cancer vs normal
http://blogs.nature.com/news/2012/09/fighting-about-encode-and-junk.html
http://blogs.discovermagazine.com/notrocketscience/2012/09/05/encode-the-rough-guide-to-the-human-genome/
Applications
• Visible as genome tracks in UCSC
• Mutation from
– Cancer sequencing
– GWAS
– Find out what that part of the genome is doing
• Compare with your cancer data (RNA-seq)
• Comparative genome analysis
• Gene or pathway of interest
Online Resources
• Interactive graphics in online version of paper
• Interactive app on Nature ENCODE main page
www.nature.com/encode/