Term
| What's important about the activation domain of Gal4p? |
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Definition
| It can be attacghed to DNA binding domain of any other DNA binding protein by using simple molecular cloning methods i.e LexA |
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Term
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Definition
| brings activation domain in proximity to the promoter |
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Term
| Characteristics of Gal4p activation domain? |
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Definition
acidic (large # of Aspartic and glutamic acid residues), hydrophobic residues help in protein-protein interactions
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Term
| Purpose of transcriptional activation domain? |
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Definition
| regulated recruitment of proteins to promoter |
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Term
| What is a mediator complex? |
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Definition
| a coactivator, a bridge between TF's and RNA Pol |
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Term
| What is a chromatin remodeler? |
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Definition
| shifts nucleosomes around to make room for proteins binding DNA. It can be recruited by TF's |
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Term
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Definition
| Histone acetyl Transferase, acetylates nucleosomes to aid chromatin remodelers |
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Term
| Explain an experiment we could use to observe the function if an activation domain? |
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Definition
- attach cloned template DNA to a chromatography column.
- Add the sequence specific TF containing activation domain & allow to bind.
- Add other TF's and allow to bind.
- Wash unbound proteins
- Add NTP's and allow transcription
- Analyze transcripts by primer extension etc. and note which TF;s have to be bound to make the desired transcript
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Term
| What did Lin and Green do? |
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Definition
determined how Gal4 Acidic activation domain functions. - created a plasmid with Galf4 binding & dimerization domain and added an artificial acidic activation domain.
- made a lot of proteins in e.coli, broke the cells and purified the extracted proteins.
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Term
| Explain the findings of Lin and Green's experiment |
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Definition
- they had lanes with bands and added GAL4 before and after washing & w/out washing.
- Gal4 and HeLa nuclear extract alone contain all factors for high level transcription.
- PIC can't stably bind to template w/out Gal4-AH
- Gal4-AH promotes PIC formation
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Term
Explain the missing factor experiment for Gal4-AH function and its findings? |
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Definition
Added factors individually +/- Gal4-AH then wash, then added back missing factors not included in first step. - only TFIID can bind w/out Gal4-AH, but TFIIB and therefore Pol II binding requires Gal4-AH
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Term
| What is a supressor mutation? |
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Definition
| second site mutations that reverse or alleviate the effects of a previous mutation. |
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Term
| What is intragenic supression? |
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Definition
| second site mutation is within the same gene as the first mutation |
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Term
| What is an Intergenic supressor? |
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Definition
| Second site mutation is in different gene |
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Term
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Definition
| Pol II bound to mediator complex |
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Term
| Explain the Activator Bypass experiment. |
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Definition
By using an intergenic supressor mutation in mediator complex, Gal11"P"; the Asn342Ile mutation can bind to Gal4 DNA binding-dimerization domain. Galf 11 and Gal 4 are essentially swapped intergenically and we no longer need the acivation domain b/c of this mutation. |
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Term
| True or False. Transcription occurs on free DNA. |
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Definition
False, Transcription occurs on nucleosomal templates. |
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Term
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Definition
| consists of DNA coiled around histones; any of the repeating subunits of chromatin. |
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Term
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Definition
Spt-Ada-Gcn5-Acetytransferase a coactivator helps facilitate binding of TBP to promoter but does bind to DNA itself. - Contains Gcn5, a histone acetyltransferase
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Term
| Explain the use of Chromatin Immuniprecipitation (ChIP)? |
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Definition
a powerful tool to determine the sies along a chromosome where interactions w/ specific proteins occurs in vivo. |
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Term
| Explain what it means to sonicate, in what type of analysis is this used? |
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Definition
| High frequency waves break up DNa into small pieces b/w nucleosomes- to allow studying of the sequences a protein was bound to. Used in ChIP. |
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Term
| What does Formaldehyde do in ChIP? |
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Definition
| Cross-links amino groups of proteins close to one another and DNA. |
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Term
| Explain the steps in Chromatin Immunoprecipitation? |
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Definition
- Formaldehyde cross-links cells via protein-protein or protein-DN A
- Lyse the cells then sonicate the lysate to get smaller fragments.
- Add antibody (or epitope tag) for protein of interest --> the DNA sequence associated with the protein will be selected.
- Sample washed, then heated to reverse cross-link
- Analysis by PCR, microarray, or direct hybridization
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Term
| Explain the ChIP method and results using antibody to HA-tagged Brf1p? |
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Definition
- Create yeast strain with HA-epitope tagged BRF1 gene
- Crosslink w/ formaldehyde
- Lyse cells, sonicate chromatin
- Precipitate Brf1-bound chromatin w/ anti-HA antibody
- AnalyzeDNa by PCR
just as hypothesized Brf1p is part of TFIIIB complex and so it should only be present at all tRNA gnes, and not present in other genes.
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Term
| Explain the findings of ChIP analysis of GAL1-10 promoter region upon switching cells to galactose? |
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Definition
SAGA complex is reqrd for induction of GAL genes when switching cells to galactose. They tagged the Spt3p and Spt20p proteins of the complex. Both of the proteins were found to be predominantly in UAS region onyl when cells are grown on galactose--> Gal4p acitvation domain recruits SAGA to UAS
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Term
| What is Spt3p significance to Gal gene. |
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Definition
Spt3p directly interacts with TBP, mutations in this inhibit galactose induction of Gal genes. Mutant (SPT15=TBP) cannot bind TATA box or induce GAL gene. A supressor mutation will cause an even stronger interaction. spt3-401 restores TBP recruitment & Spt15-21 creates an even stronger Spt3p-TBP interaction |
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Term
| What is order of protein binding to GAL 1 promoter? |
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Definition
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Term
| What are the different possible mechanisms for activating transcription? |
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Definition
- Recruitment of TBP by Spt3p
- SAGA Gcn5p (HAT) may acetylate local histones and promoters; but not reqrd for Gal1, reqrd. for other SAGA genes.
- Possible recruitment of TFIIB and TAFs
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Term
| What is the major region we discussed when talking about the human β globin gene? What is the major characteristic of this region? |
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Definition
| human β-like globin genes have a large enhancer complex called the LCR (Locus Control Region). This region is characterized by DNase I hypersensitive sites. |
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Term
| Name the 3 levels of globin gene transcriptional regulation. |
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Definition
- Tissue Specificity- genes transcribed only in erythroid cells
- High Level of transription-RBC's have 90% hemoglobin, so globin mRNA is predominant mRNA in differentiating RBC's
- Developmental Stage Specificity- Different globin genes are transcribed at different stages of embryonic development through birth.
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Term
| What globin genes are turned on in the fetus, the adult? |
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Definition
Gγ & Aγ in the fetus b/c they have high affinity for oxygen to pull oxygen from mother Adults have mainly β globin |
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Term
| Explain briefly what types of TF's bind and where to when discussing 3 levels of globin gene regulation |
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Definition
Tissue Specificity is determined by TF's bound to LCR and promoters High Level of transription- TF's bound to LCR
Developmental Stage Specificity- stage-specific TF's bound mainly at the promoters |
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Term
| Define DNase I Hypersensitivity (HS) |
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Definition
| Chromosome regions that have few nucleosomes are readily attacked. Active genes generally show hypersensitivity at promoters and enhancers |
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Term
| What kind of experiment can we do to find DNase I HS sites? |
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Definition
- Isolate nuclei from cells
- Add low conct. of DNase I (endonuclease), sites w/out nucleosomes will be preferentially cut.
- Purify DNA away from all chromatin proteins (SDS, proteinase K, phenol/chloroform)
- Digest DNA w/ restriction enzyme to make even smaller fragments that will be easier to resolve.
- Resolve DNA on agarose gel and detect size by Southern blotting.
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Term
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Definition
Early replicating transcriptionally active hyperacetylated (highly) histone tails which are less condensed ("open). more sensitive to nucleases. Euchromatin is characteristic of erythroid cells maintained by the LCR |
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Term
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Definition
late replicating, transcriptionally repressed hypoacetylated (low) histones in a more condensed, "closed" structure. Characteristic of non-erythroid cells (not transcribed) and mutant erythroid cells (poorly transcribed) |
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Term
| What is characteristic of the disease Hispanic Thalassemia |
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Definition
have chromosomal deletion that removes most of the LCR from one copy of chromosome 11--->no transcription of globin mRNA. Without the LCR, rest of the HS sites do not form, regions of chromosome that should be eychromatic are actually heterochromatic. |
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Term
| what's Somatic cell fusion? |
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Definition
| fused cells from Hispanic thal patients to mouse erythroleukemia cells. |
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Term
| Describe how we would Produce Transgenic mice? |
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Definition
- Dlone DNA fragment containing gene of interest and promoter and enhancer regulatory element
- Microinject DNA solution into male pronucleus of fertilized mouse embryo... integrates randomly into mouse chromosome and can be observed through development.
- Implant manipulated embryo into pseudopregnant female surrogate
- Analyze genotype and phenotype of live pups.
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Term
| What is the key difference of transgenic experiment in mice compared to yeast? |
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Definition
linear DNA inserted into metazoan cells, will likely not have homologous recombination. The more likely scenario is non-homologous end joining where ends are directly ligated randomly into DNA w/out concern for homology and multiple copies may be inserted into same site. |
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Term
| As discussed transgenic mice will randomly integrate DNA into chromosomes, many will integrate into heterochromatic site and no trancription. how can we override heterochromatin in erythroid cells? |
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Definition
| We have to gene to LCR sequences to override |
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Term
| What are the 4 types of transcription factor binding sites found adjacent to DNase HS sites, what kind of motifs do they bind to? |
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Definition
Binding of TF's creates euchromatic regions only in erythroid cells. GATA-1-2- (GATA family zinc fingers) EKLF/KLFs- (Cys2-His2 zinc fingers) SP1/GC rich BPs- (Cys2-His2 zinc fingers) NF-E2/AP-1-like (bZip homodimers and heterodimers) |
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Term
How did they find where enhancer function was lost in the β-globin gene?
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Definition
They made transgenic mice containing smaller and samller piecs of HS2 region attached to β-globin gene. They found that deletion of tandem NF-E2 sites (ΔAP1) results in only 2% expression. |
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Term
| at what time and what genes do the different stages of mice development express? |
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Definition
- Yolk Sac- 11.5 days εY+βh1
- Fetal Liver- 16 days βmaj+βmin (60/40)
- Birth- 21 days βmaj+βmin (80/20)
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Term
| Which globin genes coinicide in mice and humans? |
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Definition
Human γ-globin & mouse fetal globin βh1 Human β-globin & mouse adult βmajor |
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Term
| What is active transcription dependent on and what is proper developmental expression dependent on? |
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Definition
activated transcription of globin genes dependent on inclusion of LCR Proper developmental expression reqrs. β and γ |
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Term
| "Stage-specific" TF's involved in both activation of proper gene and repression of transcription of other genes not expressed, give 2 examples. |
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Definition
EKLF- erythroid Kruppe-like factor, reqrd for β-globin transcription but not for γ or ε. Binds CCACACCCT GATA-1- first erythroid-specific transcription factor identified, reqrd. for erythroid cell maturation. Binds WGATAR |
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Term
| Explain the initial step in cloning Transcription factors? |
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Definition
Natural mutations, linker scanning mutagenesis, transcriptional analysis and DNA footprinting to identify enhancer and promoter sequences important in transcriptional analysis. Now we can use this info to clone the gene for the protein that binds to the sequence.
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Term
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Definition
After we identify our protein we... - Affinity Purification- attach synthetic oligos for TF to chromatography support, incubate w/ nuclear extract. Bound protein eluted and purified.
- λ phage gt11 library screening- allows direct cloning of cDNAs of binding proteins under lac promoter. Produce phage packaged with head filled with library DNA. Produce plaques then filter probe.
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Term
| What did Linker scanning mutagenesis, sequence comparisons and natural mutations do in identifying binding of EKLF? |
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Definition
Identified "CACCC" box in both β & γ globin promoters. Extended sequences in gel shift experiments showed that EKLF bound to β promoter CCACACCCTwith 8 times higher affinity than it bound to γ promoter CTCCACCCA |
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Term
| Describe Reporter gene assays? |
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Definition
| clone promoter and other regulatory sequences (enhancers) of gene adjacent to a heterologous reporter gene (gene not present in organism being studied, and product which is easily detectable) and measure level of protein produced by that gene. |
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Term
| Name some typical Reporter genes and their functions. |
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Definition
CAT- chloramphenicol acetyl transferase- measures the attachment of radioactive acetate to cloramphenicol. E. Coli β galactosidase- (lacZ gene)- colorimetric assay of chromophore-galactose conjugate; produces colored product when cleaved by β-gal. Firefly Luciferase- reacts w/ substrates luciferin and ATP to emit visible light GFP- directly detect emitted light
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Term
| What will Reporter gene assay show us in terms of EKLF? |
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Definition
plasmids contain part of LCR, gamma or beta promoter and reporter gene. Co-transform into K562 cells w/ plasmid expressing EKLF nfrom SV40 promoter. EKLF has much higher increase in beta promoter expression. proposed to be a "switching factor" when switching from gamma to Beta. |
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Term
| What will targeted deletion of genes in mouse embryonic stem cells do for us? |
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Definition
| allows for the creation of knockout genes in mice by using homologous recombination. remember homologous recombo is rare event in metazoic cells. |
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Term
| How can we observe homologous recombo products in mice? |
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Definition
Clone deisred target gene into a vector containing HSV thymidine Kinase (TK). Insert target gene in place of neo. Cut plasmid. Transform DNA into ES cells and allow homolog recomb. Collect cells and use gancyclovir to kill cells that have retained TK (random integration. We have cells enriched in homologous recombination. |
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Term
| Anemia of EKLF mutant mice is due to....? |
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Definition
defective transcription of adult mouse βmaj. EKLF -/- develop normally until develop. switch to adult β-globin gene. |
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Term
How does an LCR function? |
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Definition
β-globin LCR has multiple binding sites for multiple TF's. Only in erythroid cells will a large compelx form at these sequences and form the cahracteristic HS sites.
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Term
| What are the 4 models of globin LCR functioning at long distance? |
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Definition
- Looping model- LCR brought into proximity to the promoter by protein-protein interactions b/w promoter and LCR associated TFs and co-activators.
- Tracking Model- LCR is an "entry site" for TF's and co-activator, which then track down chromatin until reaching promoter
- Facilitated Tracking model- combo of looping and tracking. bind to LCR to form small loop then tracks to deliver LCR complex of TF's to promoter
- Linking Model- factors bind to LCR which nucleates sequential binding of other TFs along chromatin creating a propogating open chromatin domain.
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Term
| Do enhancers/LCRs really form chromatin "loops", how do we detect this?? |
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Definition
Chromosome conformation Capture (3C) analysis - Cross-link with formaldehyde to bring distal parts together.
- Digestion w/ Restriction Enzyme's so that interacting fragments are still attached
- Ligate so DNA ends are near each other
- Analyze via PCR newly proximal elements
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Term
| What did 3C analysis of mouse ß-globin locus show? |
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Definition
| There was a peak at ßmaj in adult erythroid. W/out regulators such as EKLF and formaldehyde there is significant reduction in transcription, perhaps the LCR is no brought near the promoter. |
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Term
| What is conclusion to 3C analysis of mice ß-globin gene in mice? |
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Definition
| Chromosomal regions form "domains" of active or inactive loops and sub-structures to help activate transcription. |
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Term
| Define Chromatin boundaries (boundary elements). |
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Definition
| think of them as walls or obstacles for enhancers. their overall function is to prevent regulator elements of one domain from influencing promoters in a different domain. include barriers and Insulators (enhancer blockers) |
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Term
distinguish between Barriers and Insulators (Enhancer blockers)
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Definition
Barriers- elements that prevent spread of heterochromatin into euchromatin domain Insulators/Enhancer blockers- prevent an enhancer from promiscuously interacting w/ inappropriate promoter when positioned in between.
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Term
| What is characteristic of the chicken ß-globin locus? |
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Definition
| Contains DNase I HS element (HS4) that functions as an insulator |
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Term
| Describe Cell culture assay for insulator function |
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Definition
Place wrong promoter and see if enhancer will activate transcription. Human-Υ for neoR w/ HS2 enhancer Transform linearized DNA where promoter and enhancer are active, DNA will randomly integrate DNA w/ non-insulator allow enhancer to function and transcribe neoR DNA w/ insulator will block enhancer ability to activate reporter gene and colonies are reduced. Adding more insulators will further decrease # of colonies. |
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Term
| Explain why they think HS4 boundary element may be multifactorial, acting as barrier and insulator? |
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Definition
| They found that HS4 region is already a promoter. CTCF What they aren't sure about is why there is a HS site here, seperates euchromatin and heterochromatin. |
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Term
| I STOPPED AT TOPIC 15 pg 7 out of 8 |
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Definition
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