Module 3-Analysis of protein-DNA interactions.pdf

Methods to study and manipulate DNA and their applications Learning objectives - 4 • Understand the biological importance of protein-DNA interactions • Get familiar with techniques to explore protein-DNA interactions • EMSA • DNAase footprinting • Pull down assays • Reporter assays • CHIP • Underst...

Methods to study and manipulate DNA and their applications Learning objectives - 4 • Understand the biological importance of protein-DNA interactions • Get familiar with techniques to explore protein-DNA interactions • EMSA • DNAase footprinting • Pull down assays • Reporter assays • CHIP • Understand the basis of gene silencing using RNAi Protein-DNA interactions • Analysis of protein-DNA interactions is central to understanding several biological processes Protein-DNA interactions DNA packaging Regulation of gene transcription transcription factors get recruited and bind DNA repair mechanisms Cells are constantly exposed to stressful environment Recruit multiple proteins around sites of DNA damage to unwind DNA, cut out pieces of damaged DNA, and fill gaps with new nucleotides/ perform legations DNA is not naked in nucleus • tightly packed by histone molecules • regulates access for gene expression (also takes up less space this way) • epigenetic regulations: how modifications on histone molecules regulate gene function Transcription factors bind and get recruited to regulatory region so that RNA polymerase can be recruited and do its job of copying the gene and making the mRNA • a lot of protein interaction has to occur on the strand for that to happen Several techniques allow to study protein-DNA interactions including DNAase footprinting, EMSA, DNA pulldown, reporter assays, and CHIP some are older than others and les used such as DNAase foot printing and EMSA Protein-DNA interactions Electrophoretic Mobility Shift Assay (EMSA) DNA+ protein + detection antibody Big molecular DNA+ proteinAdd protein to weight Based on electrophoresis technique • Used to study proteins binding to known DNA oligonucleotide probes and the affinity or specificity of the interaction. detect if there is an interaction or not Free DNA what are the proteins that can bind to this nucleotide sequence? Are there proteins that are able to recognize this specific nucleotide sequence? Are these sequences involved in regulation by transcription factors or other things or is this a site where a DNA repair enzyme is susceptible to bind? • Can be performed using a specific protein (usually a recombinant protein) or a mix of proteins (ex. A nuclear extract) • Principle: Protein–DNA complexes migrate more slowly in than free DNA molecules in an agarose gel, causing a mobility shift mass of protein will hinder mobility in gel • certify that shift was caused by the protein you think was caught in the shift shift • Adding an antibody to the binding components creates an even larger complex, which migrates even slower during electrophoresis. This is known as a “supershift”. This is used to confirm the identity of the DNA-binding protein supershift • Protein-DNA interactions Electrophoretic Mobility Shift Assay (EMSA) • An example where EMSA is used to examine the affinity and the number of binding sites between a protein (here MBNL1) and a particular nucleotide sequence (here on RNA) Increasing concentration added Start to see second band - shift additional binding sites at higher molecular weights No protein RNA interaction detected Probe Supershift isn’t in this experiment because we used rna with only MBNL1 so we are 100% sure that what we see is binding with it only Protein-DNA interactions DNAase footprinting Radiolabeled end • Detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. DNAase cleavage sites DNA binding protein Map where protein is binding on sequence Proteins are often large - hide restriction site • DNA strand is end-labeled with 32P or a nonradioactive marker. • • inclubate piece of DNA w/ DNA binding protein - if it binds it will result in protected region if it does not bind, it will remain free and not cover any restriction site on DNA • DNA is exposed to DNAase in absence or presence of protein (usually a nuclear lysate) Add restriction enzymes • compare experiment where protein was added vs. One where no protein is added and examine different banding patterns Protein-DNA interactions Biotinylated DNA pulldown assay • Pull-down assays are used to selectively extract a protein–DNA complex from a sample. This molecule specifically binds biotin on the probe has known sequence • DNA probe labeled with a high affinity tag (such as biotin) is added to sample easy to pull down with centrifugation • will pellet at bottom of tube since it is huge beads • Probe binds to transcription factors • Probe-transcription factor complexes are pulled down with beads (agarose or magnetic beads) • Transcription factor is identified using protein identification techniques including proteomics When you expose cells to interferon, STAT1 expression goes up in nucleus allows us to identify genes that are regulated by STAT1 Once recovered, unwind DNA from oligo and see what proteins or transcription factors, etc. are attached • Detects interaction but does not tell you about the function or impact of the interaction on genetic function (ex. Does it increase/decrese expression of the gene, have no effect, etc.) Protein-DNA interactions Reporter assay The luciferin/luciferase system is frequently used • Used to investigate the genetic regulatory elements that control the expression of genes of interest. • For this, the regulatory elements or interest are fused to a reporter gene to monitor the amount of the reporter protein expressed. • Reporter expression is directly correlated with the activity of the regulatory elements. • Good reporter (such as luciferase) proteins should: • Be easy to detect • Not be present endogenously in the cell type/specie studied Allows specificity - can be sure that if outcome measure shows up, it is entirely specific to interaction of promotor binding to regulatory region triggering the expression of reporter gene as this reporter gene does not normally exist in the cell Binds to regulatory element/promoter region on DNA upstream of gene they regulate expression of genes If it binds, what is this impact on expression? sequence of promoter region Shows effect of transcription factor • emits light when gene is expressed shows transcriptional activity The more light being emitted, the more active the gene expression driven by PGC1 alpha Protein-DNA interactions Chromatin Immuno-precipitation (CHIP) Used to: • Antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets. • Utilizes antibodies that selectively recognize and bind proteins, including histones, histone modifications, transcription factors, and cofactors, to provide information about chromatin states and gene transcription. • Used to investigate a particular protein-DNA interaction, several protein-DNA interactions, or more typically the interactions across the whole genome. pull down proteins, with the DNA using antibodies Ex. Acetylation and the DNA that comes w/ it opens door to study of epigenetic regulation of gene expression Amendable to large scale analysis Protein-DNA interactions Chromatin Immuno-precipitation (CHIP) 1.DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). to preserve DNA protein interactions before you shred cell open w/ centrifugation homogenization, etc. Since you might induce false interactions when you add “glue” (cross-links) mostly used to study epigenetics Histones nucleus w. DNA 2.The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. antibody binds to whatever p53 is present in DNA/ histones Soup of DNA fragments w/ histones 3. DNA fragments associated with the protein(s) of interest are immunoprecipitated using an appropriate proteinspecific antibody. Isolate these fragments high through-put sequencing 4. Associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. broad overview of what p53 is affecting in terms of gene regulation over the entire genome Map of all the genes that are regulated by p53 - either by direct binding to DNA or because it modified thehistones around and mofided the accessibility Silencing gene expression • Gene silencing is central to the study of gene function. • Gene silencing is often considered the same as gene knockdown i.e. their expression is reduced (typically above 70%) but not absent (in contrast to gene knockout) Never 100% though • Gene knockdown informs of the biological role of a given gene • Gene knockdown often reproduce the phenotype of loss of function mutations observed in genetic disorders. • • most genetic disorders in which there is loss of function, the gene is partially expressed gene knockdown effectively mimics this effect Silencing gene expression • An important method to silence gene expression is RNA interference (RNAi) Commonly used in both in vivo, in vitro, in cell culture, and also in gene therapy in patients - clinical trials making use of RNAi every cell in our body is able to do that • RNAi is a process that occurs naturally in cells to: • Protect themselves from invaders such as RNA viruses • Combat the proliferation of transposons within DNA • Post-transcriptionally regulate gene expression at the mRNA level through short interfering RNAs, microRNAs and long noncoding RNAs • Andew Fire and Craig Mello won the 2006 Nobel prize of Medicine for the discovery of RNAi Identifies and suppress/prevent the expression of viral RNA particles tiny sequences that can jump sporadically in our genome and when they insert in some places, depending on where they insert, they might causes diseases like cancer, hemophilia, etc. • drive genetic evolution but too much of it would cause harm Patrol and regulate how mRNA, which encode proteins, are translated microRNA ressembles siRNA but come from a different region of RNA transcripts, is single stranded and forms haipins. Slightly less specific than siRNAs for target mRNAs Silencing gene expression dsRNA is cleaved into small (21 bp) interfering RNAs by the enzyme DICER. Endogenous or exogenous double stranded RNA (long-non-coding RNA) Silencing RNAs • way more spedifc • control the expression of one gene usually The fragments of one of the siRNA strands (the leader strand) are integrated into a multisubunit protein called RISC (RNA-Induced Silencing Complex) RISC directs the sequence-specific silencing of the target mRNA molecule by siRNA (Viral RNA) hairpin RNA Work the same • less specific than siRNA • can bind sequences on multiple mRNAs so they will regulate the expression of multiple genes when silencing RNA binds to its complementary sequencee on messenger RNA, it will block the translation of this particular mRNA • it can also trigger the degradation of the mRNA Silencing gene expression • Methods of mediating the RNAi effect involve small interfering RNA (siRNA), short hairpin RNA (shRNA) in both cases, will knock down gene of interest but w/ some differences • SiRNA: • simple to manufacture and use • transient in duration very short sequence of nucleotides Get in SiRNA vial, put in culture media and SiRNA will go in cell and be processed through the DICER enzyme and RISC complex and generate single stranded siRNA that binds to mRNA of interest and knock down expression of gene and therefore, reduce expression of protein degraded pretty fast when they get into cell - when there is no more left, the effect vanishes and progressively, the gene will start to be re-expresed Put in plasmid and transfect cells with plasmid • ShRNA: • allow for high potency long lasting effect More robust knock down of gene over a longer period • easiest and more broadly used approach plasmid goes into nucleus and it will continuously generate ShRNA • using DICER enzyme we will produce siRNA and effectively knockdown gene expression • Less shRNA copies are required to drive the effect therefore less off-target effect More complicated to work with but it lasts longer