Biology Reference
In-Depth Information
Box 4.1 Experimental Technologies for Identifying ProteineDNA Interactions
Chromatin immunoprecipitation (ChIP), coupled with either
microarrays (ChIP-chip) or sequencing (ChIP-seq):
What it provides:
l
Advantages:
l
Does not require ChIP-grade, specific antibodies
In vivo assay
l
In vivo occupancies of a protein at genomic loci
How it works:
l
Cells are treated with a cross-linking agent (typically form-
aldehyde) and lyzed, chromatin is sheared into smaller DNA
fragments; target protein is immunoprecipitated with an
antibody specific for either the protein or a tag engineered
onto the protein; cross-links are reversed to free the DNA
bound by the protein; DNA is amplified and then either
labeled for hybridization to microarrays or prepared for
next-generation sequencing; statistical comparison to an
'input' sample (which indicates what was present before
immunoprecipitation) permits identification of significantly
'bound' regions. Controls for antibody specificity can
include a 'mock' ChIP (without antibody), ChIP in a sample
that lacks the TF of interest (i.e., in a mutant or by RNAi), or
ChIP with an independent antibody against the TF of interest
Throughput:
l
Can be used for low-abundance TFs
Disadvantages:
l
l
Spatial resolution of binding events is lower than that ach-
ieved by ChIP-seq
Temporal resolution of binding events is lower than that
achieved by ChIP-seq, since the Dam methylation event
persists after TF binding
l
Expression of Dam fusion protein needs to be optimized so
that level of expression is low and does not result in
potentially artifactual TF binding due to TF overexpression
l
A higher-throughput version of this technology using
sequencing instead of a microarray-based readout has not
yet been described
l
Protein-binding microarrays (PBMs):
What it provides:
l
High-resolution, comprehensive, in vitro DNA-binding
specificity data for either individual TFs or TF complexes
How it works:
l
Genome-scale
Low at protein level because TFs need to be immunopre-
cipitated individually
Advantages:
l
l
Protein of interest is generated by in vitro translation, puri-
fied from native cells expressing the TF, or purified from
a heterologous expression system, and then applied directly
to a double-stranded DNA microarray (the DNA is naked,
not chromatinized); the protein bound to the array is
detected with an antibody specific for either the protein or
a tag to which the protein is fused (by recombinant DNA
cloning); if the primary antibody is not fluorophore-conju-
gated, then a fluorophore-conjugated secondary antibody is
subsequently applied to the labeled, protein-bound array;
data analysis of the antibody-derived fluorescence signal
intensity compared to the DNA-derived fluorescence signal
intensity permits identification of preferred DNA-binding
sequences
Throughput:
l
Condition-specific, in vivo binding data
Disadvantages:
l
Difficulty in identifying individual TFeDNA-binding sites
within a bound region
Binding events can be due to direct or indirect TF-DNA
association
[39,44]
l
For many TFs, specific ChIP-grade antibodies that do not
cross-react with TF family members are not yet available
l
10
7
cells
Typically requires at least 5
l
Difficult to detect binding events that are rare (e.g., involve
TFs that are expressed at low levels or in just a few cells
within a population of cells)
l
Does not provide data on all possible DNA-binding sites of
TFs (i.e., condition-dependent binding)
l
Can be either genome-scale covering all genomic TF-
binding sites
[46]
, or comprehensive, synthetic sequence
design covering all possible k-mers (typically k
DNA adenine methyltransferase (Dam) fusion and profiling
(DamID):
What it provides:
l
In vivo occupancies of a protein at genomic loci
How it works:
l
ΒΌ
10 or more
bp)
[47]
l
Multiple proteins can be assayed simultaneously in a multi-
plex microarray format
Advantages:
l
Protein of interest is expressed as fusion with the Escherichia
coli DNA adenine methyltransferase (Dam) protein; wher-
ever the protein associates with DNA, Dam methylates
adenines within nearby GATC sites; methylated sites are
detected by digestion with a methyl-specific restriction
enzyme followed by amplification, labeling, and hybrid-
ization to a DNA microarray; comparison to a 'Dam alone'
control (i.e., no fusion) permits identification of significantly
methylated, and thus 'bound', regions
[45]
Throughput:
l
Provides data on DNA-binding specificity of particular
protein(s)
(e.g., TFs) under buffer conditions applied to
microarray
Multiple proteins can be assayed in parallel simultaneously
l
Tens of thousands of different sequences can be assayed in
a single PBM experiment
Disadvantages:
l
l
Impact of in vivo interactions with other proteins or mole-
cules is unknown unless specifically tested
Naked DNA on microarrays does not provide information
on protein binding to chromatinized DNA
l
Genome-scale (scale depends on genomic regions covered
by the DNA microarrays)
Suitable source of protein (full-length or DBD only) not
always obtainable
l
Proteins are fused to DamID and profiled individually
l
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