1. DNA:chromatin interactions
Exploring transcription factor binding and the epigenomic landscape
Lisa Stubbs

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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!)

8. 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

9. 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
ChIP fragments
Seq reads

10. 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

11. DNAse sensitivity assays are antibody free
The first approach: from Crawford et al., Genome Research 16:123, 2006 (Francis Collins’ laboratory)
Digest with DNAse I to “erase” all the hypersensitive regions
Easier to do– less need to optimize and minimize DNAse cutting
Polish and ligate the remaining double-strand 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

12. 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

13. Related methods and twists on the theme (see Furey et al
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:
With carrier DNA: not the best for sequence analysis but can be done
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”

14. 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

15. 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)

16. 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

17. Overview: ENCODE and modENCODE
Data paint an extremely similar picture for human, mouse and Drosophila cis-regulatory 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

18. ENCODE Chromatin profiles are displayed in the UCSC browser