Lecture 4 - Chromatin dynamics and the histone code Flashcards
Which PTM corresponds to which domain and corresponds to which chromatin state
Methylation is code for chromodomain and code for heterochromatin. Acetylation is code for bromodomain and code for euchromatin.
What drives heterochromatin formation
Trimethylation of lysine 9 on histone H3 (H3K9 -> H3K9Me3)
Steps to heterochromatin formation
1) H3K9 methyl transferase 2) HP1 chromodomain binds to H3K9Me3 3) HP1 self-oligomerizes thus cross-linking nucleosomes into more-tightly packed chromatin
Model to heterochromatin formation is called a _________________ and these can _______ and _________ histone codes
positive feedback loop, reinforce and spread
what HP1 does in addition to self-oligomerization. effect ?
Binds and recruits H3K9 methyl transferase. effect = heterochromatin tends to spread along chromosome once initiated
Euchromatin tends to ________ along the chromosome because of a __________
spread. positive feedback loop.
what H3K9 HMT stands for
H3K9 histone methyl transferase
__________ can isolate adjacent chromosomal domains and _________ them. They can separate _______ chromatin from spreading _______ and _____-coated ____________
Boundary elements. distance spread. active chromatin. silenced and HP1 coated heterochromatin
what is the evidence of the link between gene expression and chromatin structure
DNase 1 digestion
DNase 1 is an ___________. It digests _____ DNA by cleaving it only between the __________. The result is ________ of DNA that are _______ bp long. _________ chromatin is protected from DNase 1 cleavage.
endonuclease. naked. nucleosomes. fragments. 150. Condensed
Prediction in a DNase I digestion experiment
Genes should be more susceptible to DNase I digestion in cells where they are expressed
DNase I cleavage experiment was done on ___________ gene, expressed in _____________ but not in _________ cells. What is expected ?
chicken globin. 14-day erythroblasts. MSB. Globin gene expected to be cleaved into fragments when DNase I added to 14-day erythroblasts but not in MSB cells.
What does DNase I cleavage on DNA from different cell types of the same organism show ?
All genes of an organism are contained in all its cells but gene expression determines cell type (or cell type determines gene expression)
Steps of southern blotting hybridization (in general) (5 STEPS)
1) DNA cleaved w/ restriction enzymes 2) Gel electrophoresis 3) On filter paper in an alkaline solution, fragments transfered from gel to nitrocellulose using alkaline solution (capillary action) 4) Nitrocellulose map incubated with labeled DNA probe 5) Location of fragment hybridized to probe is revealed by autoradiography or fluorescent imaging
Why use southern blotting (and probes) if gel electrophoresis seperates fragments
Because when a whole genome is fragmented, there are too many fragments so you can’t resolve (identify) any particular DNA fragment as a discrete band on the gel
Why do we use a blot (replica of the gel on the nitrocellulose)
Probes do not readily diffuse into the original gel
How do you obtain a probe
cloned restriction fragment that is radiolabelled (ex: radioactive isotope of the phosphorus in phosphodiester bond)
Southern blotting with the globin gene and DNase I digestion STEPS + length of globin gene
1) Isolate chromatin 2) DNase I digestion 3) Remove proteins 4) Cut pure DNA with restriction enzyme (BamH1) (will cut fragment with the gene if the gene hasn’t been digested 5) Restriction fragments seperated by gel electrophoresis 6) Detection of globin gene (4.6 kb) by Southern blot hybridization w/ DNA probe for globin gene
You can shut down a gene by expressing a ________or a ____________ (enzymes)
methyl transferase. deacetylase
Why acetylation opens chromatin
Positive charge on lysine tail changes to neutral and it reduces its ability to interact with DNA negative phosphate backbone AND ABILITY TO INTERACT WITH ADJACENT NUCLEOSOMES.
what technique can help identify if PTMs are related to global gene activity (acetylation related to transcription and methylation related to transcription repression) and what does it use
Chromatin immunoprecipitation (ChIP) uses antibody specific to acetylated histone tail.
ChIP briefly explained by comparison
Antibody affinity chromatography (proteins bound with antibody that is specific to them hang up) but with chromatin
How can ChIP be used to determine which genes are expressed in a particular cell type
Antibody specific to acetylated N terminal histone tails binds to these. In column, you obtain fractions with these histones and their DNA. By amplifying these specific DNA sequences, you obtain multiple copies of the expressed gene.
Steps to ChIP (in ChIP where you want to amplify a gene that is expressed)
1) Isolate and shear chromatin mechanically 2) Add antibody specific for acetylated N terminal histone tail 3) Immunoprecipitate 4) Release immunoprecipitated DNA and assay by PCR
How chromatin modification works for specific gene (regulation of expression)
Transcription factors (activators or repressors) bind to enhancers or silencers (sequences) and recruit enzymes (coactivators/corepressors) that modify histone tails
What modification(s) promote(s) chromatin decondensation
Acetylation (acetyl transferase)
What modification(s) promote(s) chromatin condensation
Deacetylation (deacetylase) or H3K9 or H3K27 Methylation (methyl transferase)
All enhancers are ____ which means that they have to be physically in ______ with the gene they are affecting
cis. line
2 main steps in transcriptional control
1) (pioneer) TF binds to regulatory sequences in condensed chromatin and recruits chromatin remodelling enzymes 2) TF (other one) that will bind to mediator complex can now bind their specific sequences (that were closed before) and bind the mediator complex (goal is stable elongation ?)
REMINDER : Point of mediator is to link RNAP II to complexes that open chromatin ?
NO. HETEROCHROMATIN/EUCHROMATIN SPREADS ALONG DNA
