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Unformatted text preview: Section Notes week8 GSI: Nikki Kong Techniques Summary In Vitro:  ­ ­> DNaseI footprinting and gel shift tell you whether a protein binds to a DNA sequence or not; the former can give you the sequence of the DNA bound  ­ ­> In vitro transcription assays can tell you whether the DNA ­binding protein is a transcription activator or not In Vivo:  ­ ­> Chromatin immunoprecipitation can tell you where a protein binds to DNA in a cell because you fix their positions in the cell  ­ ­> Luciferase assay tells you whether a DNA ­binding protein is a transcription activator or repressor (dependent on basal transcription level)  ­ ­> microarrays: cDNA microarrays allow you to compare whole genome transcript levels between different samples; promoter region tiling DNA microarrays (chip) coupled with ChIP can tell you, at the whole genome level, where a protein binds  ­ ­> ChIP ­seq: tells you not only information from ChIP ­chip, but also where the protein binds in the distal regulatory regions Computer based:  ­ ­> Comparative analysis of different species can reveal evolutionarily conserved sequences, and thus potentially functionally relevant regions.  ­ ­> If you know the peptide sequence of a protein, there are softwares that help you predict the different protein motifs Luciferase Assay: You need a plasmid containing the gene that encodes for your transcription factor of interest (X), and another plasmid containing a reporter gene and a piece of DNA (usually promoter) that you know X binds to. 1. You introduce the DNA into cells by either electroporation (cells in an electrical field to open up transient pores) or transfection (mediated by cationinc lipids to neutralize the negatively charged DNA backbone) 2. Takes a few days for the cells to express X, and then for X to bind and activate/repress the transcription of luciferase reporter, then for the luciferase to be translated 3. You lyse the cells to extract the luciferase enzyme and test its activity 4. Controls can be either transfected with reporter plasmid only, or transfected with reporter plasmid + empty plasmid that doesn’t have X. Either of these will give you a basal level of transcription activity. By comparing your experimental cells (X + luciferase) to the basal level, you can figure out whether X is an activator or a repressor ChIP: antibody ­based, IP=immunoprecipitation or pulldown 1. Crosslink the cells with formaldehyde to capture any protein ­protein and protein ­DNA interactions 2. Sonication or DNase treatment to make smaller fragments of DNA 3. IP with antibody that can be conjugated to a matrix (agarose, sepharose, etc) 4. Reverse crosslink by heat to release the DNA fragments that are pulled down 5. Purify DNA and analyze by PCR (useful for a few genes with known sequences so you can design primers), deep sequencing (ChIP ­seq, unbiased sequencing of everything that is pulled down, useful for distal regulatory regions), or microarray (ChIP ­chip, where the chip contains small probes that cover all promoters of the genome, or custom ­designed) Because of cross ­linking (sometimes depends on the strength, i.e. [formaldehyde]), you can get information on where a protein that doesn’t directly bind DNA, prefers to interact with DNA through another protein. For example, you can get probe where HDACs interact with acetylated histones in a region even though HDACs don’t bind DNA directly, but histones. Transcription factor motifs: these are usually DNA binding and/or dimerization motifs; a protein will need other domains such as activation and regulatory motifs. A. Zinc finger motif: need multiple fingers to bind sequence ­specifically to DNA. B. Basic helix ­loop ­helix: forms dimers usually and have many R and K residues in the part that actually contacts DNA C. Leucine zipper (b ­Zip): a leucine residue at every 7th position in the leucine zipper domain, facilitates dimerization. Also many R/K residues in the binding domain. Because of dimerization, DNA sequences that HLH and b ­ZIP proteins prefer to bind usually have inverted symmetry (rotational symmetry) Enhancers (distal regulatory regions) and insulators: Enhancer sequences are usually bound by sequence ­specific activators that are often tissue specific or signaling dependent. They are prevented from activating genes that they are not supposed to activate by insulator sequences. Insulators are often bound by proteins such as CTCF (contains 11 zinc fingers) that can mediate looping of this enhancer to the promoter region. This way, enhancers can only interact with specific promoters, and not just any promoters nearby. Roles of nucleosomes in Trxn regulation Heterochromatin Mainly for packaging, tight interactions between histones and DNA, making many regulatory sites inaccessible to transcription factors (TATA, etc) Euchromatin Open conformation, making TATA boxes and regulatory sites available for binding by txn factors How: 1. modified histone tails A. Acetylation: neutralize the lysine ­rich and thus positively charged histone tails. Done by HATs (histone acetyltransferases) and reversed by HDACs (Histone deacetylase complexes). HDAC inhibitors have some potential as anti ­ cancer drugs since they can activate apoptotic genes that are usually repressed by HDACs in highly proliferative cancer cells B. Methylation (HMTs), phosphorylation, etc— Methylation can be interpreted as histone codes: H3K4 trimethylation= gene activation, H3K9/K27 methylation = gene repression, etc. These histone marks are recognized (“decoded”) by proteins with specific domains such as PHD finger for H3K4me3 or chromo domain for K9me3. For example, Taf3, a Tbp ­associated factor in the TFIID complex, contains a PHD finger and is thought to be recruited by H3K4me3 marks to activate gene expression by assembling the rest of the TFIID. 2. Chromatin remodeling complexes: examples are ACF and Swi/Snf complexes. These are multi ­subunit complexes that use ATP hydrolysis/helicase activity to shift nucleosomes around to either reveal or conceal enhancer regions or TATA boxes, thus regulating gene expression. If you discovered a new complex with ATPase activity that you suspect is a chromatin remodeler, you could design a DNA sequence to have restriction digest sites inside of nucleosome wrapping region (somewhat known and there’s a lot of debate on whether there’s a consensus sequence) and thus inaccessible for digestion. Upon adding the complex that you’ve discovered, if you try to cut the nucleosome ­wrapped DNA piece again and it was digested, that would suggest that the nucleosomes have shifted and maybe the complex does have remodeling activity. Note: once methylation or acetylation marks are deposited, they tend to spread within a region. To guarantee region ­ and gene ­specificity of gene expression, there are often boundary elements in the gene to prevent the spreading of a particular mark to its neighboring gene that should not be regulated together. Transcription initiation revisited: 1. Chromatin remodeling by complexes such as ACF to open up the chromatin and reveal regulatory regions 2. Activating histone marks such as acetylation or H3K4 tri ­methylations are deposited by HATs or HMTs, which are recruited by other sequence ­specific activators. 3. Co ­activators recognize histone marks and recruit pre ­initiation complex with TFIID and Pol II and other TFII factors. 4. TFIIH opens up the promoter and phosphorylates Pol II CTD at Ser5 so promoter clearance can take place 5. P ­TEFb needs to phosphorylate Pol II CTD at Ser 2 for elongation to occur. But elongation itself is also heavily controlled, see below Transcription Elongation P ­TEFb (positive ­transcription elongation factor b) not only phosphorylates the Pol II Ser2, it also phosphorylates NELF (negative elongation factor). NELF and another factor called DSIF bind to the paused Pol II after promoter clearance (this step is necessary for capping enzymes to put a 5’ cap on the nascent mRNA). After both are phosphorylated (both mediated by P ­TEFb with the help of a host of other factors such as TFIIS and FACT), NELF unbinds Pol II and DSIF switches from a negative elongation factor to a positive one. Then productive elongation can continue A lot of what we know about txn elongation comes from studying HIV ­1 virus that utilizes the host txn machinery to replicate and infect other cells: TAT (trans ­activator of transcription) is an HIV protein that is synthesized early upon infection, before proviral DNA integrates even. Even though HIV relies on the host transcription machinery, the HIV promoter in the 5’ LTR is very weak for the Pol II, which always pauses after 70~80nts of the proviral DNA have been transcribed. But this nascent mRNA contains TAR which forms a hairpin structure. TAT and TAR (both HIV components) can then recruit P ­TEFb to the paused Pol II to phosphorylate CTD Ser2 and NELF for productive elongation to continue. This is an example of a positive feedback loop, since TAT is originally present at very low levels since very little mRNA is synthesized in the host cell after infection, but it can induce the production of many more mRNA by stimulating transcription elongation using the host factors and HIV’s own TAR. ...
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