W6 Methods to study protein-DNA Interactions Flashcards
what are the biological roles of protein-DNA interactions
transcription regulation
chromosome maintenance
dna replication
dna recombination and repair
methods to detect protein-DNA interactions in vitro (outside of cell)
dna footprinting
electrophoretic mobility shift assay (EMSA)
protein-binding microarray
methods to characterise or quantify protein-dan interactions in vivo (inside cell)
reporter gene assays
chromatin immunoprecipitation (ChIP)
chromosome conformation capture (3C)
basic principle of dna footprinting
protection of protein-bound dna from degradation
chemical or enzymatic digestion of free and protein bound-dna oligomers followed by gel electrophoresis
modification is used to localise the contact are between protein and dna
uses of dna footprinting
characterise transcription factor binding to regulatory dna
assess binding strength of a protein to region of dna
identity functional genes present in the human genome
different cleavage agents that can be used
most common: DNase I (cuts dna liberally but only where proteins are not bound to dna > limited quantity used to create a single nick per fragment)
others: dimethyl sulfate protection, UV irradiation
steps involved in Dnase I footprinting assay
- dna prove is radiolabelled on one end and single strand
- incubation of labelled probe with dna binding protein
- DNase I added to partially digest dna fragments > create range of fragments which differ from one another by a single nucleotide
- dna is purified and analysed by electrophoresis using a denaturing polyacrylamide gel
what is EMSA
rapid and sensitive method to detect protein-dna interactions through changes in electrophoretic mobility on a gel
principle:
- dna + protein > migrate slower on gel
- unbound dna > migrates faster on gel
gel used must be non denaturing, unlike the one used for footprinting
key features of EMSA
end-labelled (32P or biotin) oligonucleotide probe
purified protein or complex mixture (WCL or nuclear extracts)
competitor dna (nonspecific or specific)
non-denaturing polyacrylamide or agarose gel electrophoresis
how to design and prepare dna probes for EMSA
two complementary ssODN annealed to generate dsODN, usually 20-25 bp
dna probes can be labelled with radioisotopes or fluorophores
short nucleic acids are easily synthesised and contain small number of nonspecific proteins binding sites
preparation of protein extracts
can be prepared from whole cells or isolated nuclei
nuclear extracts results in isolation of only binding factors with access to dna
whole cell extracts enables entire dna binding protein content of cell to be examined
dna binding conditions for EMSA
dna probe interactions sensitive to mono and divalent salt concentrations and pH
sequence specific interactions stabilised by relatively low ionic strength of electrophoresis buffer
soluble buffers: Tris based etc
additives to be added for EMSA
small neutral solutes like glycerol or sucrose to stabilise labile proteins
carrier protein like BSA to minimise non specific losses of binding proteins
protease, nuclease and phosphatase inhibitors
competitive vs non competitor competitor nucleic acid (unlabelled)
non specific:
- reduce binding of secondary proteins to labelled target
- protein of interest binds to target nucleic acid with greater affinity than it binds to competitor
specific: 200-fold molar excess of unbaked dna target which out-compete specific protein-dan interactions with radio labelled probe
difference between shift and supershift
to determine dna binding specificity: use competition assays > eliminates a specific shift (band not visualised if its specific binding)
to determine identity of protein: add antibodies specific to protein of interest > super shift (a higher band)
uses of EMSA
determine dna regions bound by specific transcription factors
deduce binding parameters and relative affinities of a protein for one or more dna sites
compare the affinities of different protein for the same sites
study higher order complexes containing several proteins
common reporter genes for reporter gene assays
gfp
luciferase (luc)
beta galactosidase (lacZ)
how can luciferase reporter assay be used to quantify activity of trans-acting proteins
- regulatory dna element cloned upstream of luciferase reporter gene in an expression vector
- reporter construct is transferred into cells together with transcription factor of interest by transfection
- enzymatic activity of luciferase is measured using luminometer
applications of reporter gene assay
monitor the transcription of specific genes in cells
evaluate strength of promoters and enhancers
characterise the function of transcription factors or cofactors
what is chromatin immunoprecipitation (ChIP)
method to identify protein-dan interactions in vivo
captures protein-dna interactions via in vivo crosslinking
quantity of dna bound to protein of interest can be measured via conventional pcr or qpcr
applications of ChIP
detect specific protein-dan interactions in vivo
identify multiple proteins associated with a specific genomic locus
identify different regions of the genome associated with a particular protein
determine relative density of factors along genes or genome wide
procedure of ChIP
- fixation: in vivo cross linking or proteins to dna using reversible crosslinkers
- cell lysis and isolation of chromatin
- chromatin fragmentation by sonication of enzymatic digestion
- immnunoprecipitation of protein-dna complexes
- reversal of cross linking and isolation of dna
what is ChIP-seq
chromatin immunoprecipitation followed by sequencing
dna fragments associated with protein target are sequenced directly
advantages of ChIP-seq
higher resolution
fewer artefacts
greater coverage
larger dynamic range
