DNA:chromatin interactions
Exploring transcription factor binding
and the epigenomic landscape
Lisa Stubbs
Eukaryotic genomes are complex structures comprised of
modified and unmodified DNA, RNA and many types of
interacting proteins
• Most DNA is wrapped around a “histone core”, to form nucleosomes
• The classical histone protein complexes bind very tightly to DNA and prevent
association with other proteins
• Modifications of the classical histones, or their replacement with unusual histone
types under certain conditions, can “loosen” the interaction with DNA, allowing
access to transcription factors, RNA polymerase, and other proteins
All four histones in the tetramer have “tails” that can
be modified in various ways, but the most
consequential modifications, with respect to
transcriptional activity, appear to involve methylation
or acetylation of Lysines (K) in histone H3
Histone H3 modifications, especially methylation and
Acetylation, mark “open” or “closed” DNA
• CLOSED: Histones bound more tightly to DNA
– H3K27Me3, H3K9Me3
• OPEN: Histones can be displaced by TFs, RNA Polymerase, and
other proteins
– H2K27Ac, HeK4me1, H3K4me3
• Histone marks, together with other assays of open chromatin,
are presently the only reliable indicators of the locations and
activities of regulatory elements
Many types of regulatory elements
• “Docking sites” for site-specific regulatory proteins
– Transcription factors, TATA binding factors, and other site-specific binders
– Recruit additional proteins: co-factors, RNA polymerase and others
• Enhancers
– Tissue-specific activators of transcription
– Binding sites for proteins that interact with the promoter to enhance transcription
• Silencers
– Also prevalent, but more difficult to detect and assay
– Many transcription factors repress, rather than enhance, gene expression
– “Enhancers” and “Silencers” are not mutually exclusive! Most regulatory elements can
serve either function, depending on the proteins bound at a particular time
• Insulators
– “boundary elements” that shield genes from the enhancers or heterochromatin proteins
in neighboring gene “territories”
– Involved in establishing loop structures that isolate genes
How to find them? Chromatin ImmunoPrecipitation (ChIP)
•
Antibody to a DNA binding protein is
used to “fish out” DNA bound to the
protein in a living cell
– DNA and protein are crosslinked in the cell
using brief treatment with low
concentration of high quality formaldehyde
– Crosslinked chromatin is sheared, usually by
sonication, to yield short fragments of
DNA+protein complexes
– Antibody to a TF or other binding protein
used to fish out fragments containing that
DNA binding protein
– DNA is then “released” and can be analyzed
by various methods:
• Original method is PCR: query for
enrichment of specific (known or
suspected) DNA binding regions in
ChIP-enriched DNA
• Creates a pool of sequences highly
enriched in binding sites for a
particular protein
– Requires availability of excellent
antibodies that can detect the protein in
its in vivo context
ChIP can be used to map DNA:protein interactions of
virtually any type
• Histone modifications:
• Secondary interactions (no direct linkage to DNA)
– Histone modifying proteins, such as SWI/SNF, histone deacetylases,
histone methylases
– Cofactors that bind to TFs at particular sites, and that stablize
chromatin loops
– Proteins that link chromatin to nuclear matrix
• RNA polymerase and elongation factors, to find promoters
and active sites of transcription
• Proteins involved in DNA recombination, repair, and
replication
• All of these methods require highly specific and efficient
antibodies (which are rare!)
ChIP Analytical challenges
• Genomic neighborhoods
– Shear efficiency is not really “random”
• Some genomic regions are fragile and sensitive
• Some regions are protected from shear or degradation
– “Sticky” chromatin
• Some DNA regions bind to any antibody you use
• Chromatin-matched, co-sheared controls are essential, but mock ChIP
(with IgG treatment) is helpful too
– Other artifacts
• Centromeres: repeat sequences that are not all represented in the genome
sequence build
• Polymorphic regions, and e.g. regions that are amplified in cell line DNA
• Repeats: most programs cannot manage sequence reads that are not mapped
uniquely
• Peak width
– Transcription factors are typically sharp peaks; chromatin marks are more
diffuse
• The best tools permit the user to modify these parameters
– MACS ( Xiaole Liu Lab; Zhang et al, 2008; Feng et al. Nature Methods
2102) is a user-friendly and widely used tool
ChIP computational issues
• First step is to map reads:
BOWTIE, Novalign, BWA or other
• ChIP seq reads surround but may
not contain the DNA binding site
•
Sequence is generated from the ends
of randomly sheared fragments,
which overlap at the protein binding
site
• Gives rise to two adjacent sets of
read peaks separated by ~ 2X
fragment length
• Defines a “shift” distance between
read peaks at which you will find
the true ChIP peak summit
• Programs like MACS
automatically subtracts your
control (genomic input) from
sample reads to define a final set
of peaks
Binding site
Seq reads
ChIP fragments
Traditional methods fail with broad, flat peaks
• Most tools designed for TF proteins: discreet, sharp peaks
• Certain chromatin proteins, and modified histones in certain regions, bind
continuously to large regions of chromatin and do not yield “peaks”
• MACS in default mode will carve the “mesa” into many peaks, or not
detect it at all
• New settings in MACS 2 can be set to overcome this problem
• Other tools, e.g. Zinba (R-based) are designed specifically for this problem
Scale
chr7:
HOXA3
HOXA3
Enhanced H3K27Ac
Layered H3K4Me1
Layered H3K4Me3
hg18
20 kb
27,140,000 27,150,000 27,160,000 27,170,000 27,180,000 27,190,000 27,200,000 27,210,000
RefSeq Genes
HOXA-AS3
HOXA-AS4
HOXA11-AS
HOTTIP
HOXA4
HOXA5
HOXA7
MIR196B
HOXA11
HOXA13
HOXA6
HOXA9
HOXA-AS3
HOXA10
HOXA10-HOXA9
HOXA10
ENCODE Enhancer- and Promoter-Associated Histone Mark (H3K27Ac) on 8 Cell Lines
ENCODE Enhancer- and Promoter-Associated Histone Mark (H3K4Me1) on 8 Cell Lines
ENCODE Promoter-Associated Histone Mark (H3K4Me3) on 9 Cell Lines
NHGRI Catalog of Published Genome-Wide Association Studies
DNAse sensitivity assays are antibody free
The first approach: from Crawford et al., Genome
Research 16:123, 2006 (Francis Collins’ laboratory)
1.
Digest with DNAse I to “erase” all the
hypersensitive regions
–
2.
3.
4.
5.
6.
Easier to do– less need to optimize and
minimize DNAse cutting
Polish and ligate the remaining doublestrand ends
Ligate 5’-biotinylated linkers to the DS
ends
Shear (sonicate) or restriction-digest DNA
into smaller fragments
Purify end sequences on a streptavidin
column
Release sequences, add new linkers, and
sequence
–
Does not allow footprinting, because TF binding
sites inside the HS regions have been digested
away
Latest (and better) approach: sequences DNAse sensitive regions
per se and permits transcription factor “Footprinting”
• The easiest method uses
low concentrations of
Dnase I to generate short
fragments at sensitive
(“open) sites
• Released fragments can be
blunt-ended, ligated to
linkers and sequenced
directly
• Permits DNase
Footprinting: Very deep
sequencing can “see” short
protected regions that are
absent from the released
DNA, and appear as
protected “valleys” inside
the DNAse sensitive peaks
– protected from DNAse I
because they are occupied by
TF proteins
Related methods and twists on the theme
(see Furey et al., 2012 for review)
• Exo-ChIP
– Follows sonication with an exonuclease step, to “pare back” all but the
protein-protected region in ChIP
• Nano-ChIP
– ChIP normally required ~107 cells as input; hard to achieve for many cell types
– Nano ChIP can be carried out in two ways:
1. With carrier DNA: not the best for sequence analysis but can be done
2. Amplification after ChIP: very tricky because it can cause serious biases and
artifacts, but can be done with care; linear amplification is the best strategy
• FAIRE: formaldehyde assisted isolation of regulatory elements
– Takes advantage of the fact that open chromatin regions are hypersensitive
also to shearing and chromatin prep steps
– Basically, analyzes the input chromatin from a ChIP experiment: no antibodies
required
– Sensistive regions are digested away, creating a “valley” while protected
regions are retained and create a “peak”
Lessons from ENCODE chromatin assays: human data
• Massive deep-sequencing of multiple chromatin features in cell lines
(ENCODE), primary cell types and tissues (Epigenetics Roadmap)
• Histone H3 modifications: highlight on H3K4me1, H3K4me3, H3K27Ac,
H3K27me3
• Other chromatin proteins: e.g. P300 (acetyltransferase)
– H3K4me3 marks are enriched at active promoters
• H3K4me3 marks are largely the same in all cell lines, with a small fraction of marks
being cell-specific
– P300, and H3K4me1 without H3K4me3 is enriched at enhancers
• Most P300 peaks also contain H3K4 me1
• P300, H3K4me1 marks are highly cell-type specific
• Most P300 marks are enhancers, but not all enhancers have P300
• Most enhancers have an H3K4me1 mark but, not all H3K4me1 marks are in
enhancers
– Other marks: H3K27Ac or H3K27me3
• Mutually exclusive marks for open (Ac) versus closed (Me3) chromatin regions
• H3K27Ac is perhaps the most general mark of open chromatin: promoters and
enhancers
• Can be found in combination with H3K4 me1/me3
Combinatorial marks define subclasses of enhancers
• H3K4me1+ , H3K27Ac + mark enhancers with highest levels of activity
– Represent cell-type specific active enhancers in differentiated cells
– Mouse enhancers: gain K27Ac upon differentiation in mouse ES cells, leading to higher
expression
• K4me1+, K27Ac- marks
– Called “intermediate” enhancers, linked to a variety of non-specific cellular functions
• In humans especially, K4me1+, K27me3+ are called “poised” enhancers,
– K27me3 is a mark of polycomb repression; polycomb proteins are also associated with
these sites
– K9me3+ marks also found at poised enhancers
– These enhancers are associated specifically with development-related functions;
K27me3 may be replaced by K27Ac as differentiation progresses
– Poised enhancers are more likely to be conserved between species, and therefore most
of the enhancers that have been tested so far are probably of this subclass
• Explains why K4me1 does not always find active enhancers (finds the
“poised’ ones too)
Other properties of human enhancers
• A subset of human enhancers have been shown to give rise to non-coding
RNA
– ChIP with the RNA pol2 antibody identifies binding to enhancers that are far from any
known gene promoters
• Do not have marks that are shared by other types of promoters (e.g. H3K4me3)
• Some are verified enhancer loci, e.g. the beta-globin control region gives rise to a regulatory
RNA
• Histone marks other than H3K4 and H3K27 are also found
– For example, H2 variant H2AZ and H3 variant, H3.3
– Double variant (H2AZ/H3.3) marks are common at enhancers
– These two histone marks associate weakly with DNA and may be stripped
away by most usual treatments (e.g. low salt)
– This alters the view that “open chromatin” is fully depleted of nucleosomes;
rather, it suggests the presence of different types of nucleosomes that are
more loosely bound to DNA
• More sites of open chromatin (e.g. DNAse sensitive) exist that have
not been associated with any specific protein, implying that the
story is still more complicated
Overview: ENCODE and modENCODE
•
•
•
•
•
•
•
Data paint an extremely similar picture for human, mouse and Drosophila cisregulatory landscapes
Promoters marked by H3K4Me3
Active enhancers marked by H3K4me1 + H3K27Ac and p300/CBP
– Major difference is that fewer fly enhancers are found far from a TSS
“Poised” enhancers marked by H3K4me1+ H3K27me3 : a mix of activating and
repressing marks, waiting to be transferred to one or the other states
– Enriched in developmentally-active transcription factor and signaling
genes
Repressed regions marked by H3K9me3 (stable), K3K27me3 (dynamic)
– Active or repressive marks correlate very well with expression levels in a
particular cell
Insulators marked by CTCF and centrosomal/cytoskeletal proteins (CP190,
cohesins)
TFBS, chromatin marks and expression data can be used to predict regulatory
relationships, but the linkage between regulatory elements and “target genes”
is very hard to decipher, especially in mammalian genomes
modENCODE Chromatin profiles are displayed in the
UCSC browser
Scale
chr3R:
Spliced ESTs
10 kb
26,675,000
26,680,000
D. melanogaster ESTs That Have Been Spliced
dm3
26,685,000
FlyBase Protein-Coding Genes
tll
BDTNP Chromatin Accessibility (DNase) Stage 5, Replicate 1
BDTNP Chromatin Accessibility (DNase) Stage 9, Replicate 1
BDTNP Chromatin Accessibility (DNase) Stage 10, Replicate 1
BDTNP Chromatin Accessibility (DNase) Stage 11, Replicate 1
BDTNP Chromatin Accessibility (DNase) Stage 14, Replicate 1
BDTNP ChIP/chip: bicoid (bcd) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: caudal (cad) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: giant (gt) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: hunchback (hb) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: hunchback (hb) antibody 1, stage 9 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: knirps (kni) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: Kruppel (Kr) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: huckebein (hkb) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: tailless (tll) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: Dichaete (D) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: fushi tarazu (ftz) antibody 3, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: hairy (h) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: paired (prd) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: runt (run) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: sloppy paired 1 (slp1) antibody 1, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: daughterless (da) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: dorsal (dl) antibody 3, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: Mothers against dpp (mad) antibody 2, stage 4-5 embryos, False Disc. Rate (FDR) 1%
BDTNP ChIP/chip: Medea (med) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: Medea (med) antibody 2, stage 10 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: Medea (med) antibody 2, stage 14 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: schnurri (shn) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: snail (sna) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: twist (twi) antibody 2, stage 4-5 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: zeste (z) antibody 2, stage 11 embryos, False Discovery Rate (FDR) 1%
BDTNP ChIP/chip: RNA Polymerase II (PolII) antibody, stage 4-5 embryos, False Discovery Rate (FDR) 1%