lec 12 Flashcards
how genomes exist during interphase
chromatins
how do genomes exist during cell division
chromosomes
what are chromosomes
condensed chromatins which get duplicated before cell division
what are chromatids
sister duplicate chromosomes
euchromatin
lighter in color because it’s less condensed
Contains actvely transcribed genomic regions
Heterochromatin
darker because it’s more condensed
Includes centromeres and telomeres which are not actively transcribed
how does chromatin structure affect the transcription activity of genes?
Less condensed chromatin (euchromatin) is more active and transcribed more readily than the more condensed heterochromatin
what is the chromatin structural unit
nucleosomes
what does Metaphase chromosome look like
like X (the way you picture a chromosome) on the scale of 1400 nm
chromosome at 700 nm
condensed scaffold-associated chromatin which is all loopy with a scaffold backbone (see at 300 nm)
interphase chromosome
extended scaffold associated chromatin
what does a zoomed in chromatin fiber look like
packed nucleosomes (little balls packed together)
what do zoomed in packed nucleosomes look like
“beads on a string” basically beads with string looped around each
what is the string portion of ‘beads on a string’ nucleosomes
double helix DNA
what are nucleosomes
DNA histone particles
147 bp DNA wrapped around 8 core histones
what are histones
basic proteins rich is arg and lys (basic binds tightly to (-) charged DNA)
what are the core histones
H2A, H2B, H3, H4 (11-15 kD) each is a protein chunk which is a part of the ‘bead’
What connects nucleosomes?
50 bp linker DNA and linker histones H1 (~21 kD)
what does chromatin structure explain
where DNA hangs out inside the nucleus
what does the dsDNA in chromosomes consist of
gene families, simple seq DNA, spacer DNA, and introns
What affects the position of nucleosomes?
The number of linker histone H1, histone modification, and chromosome remodeling proteins
What allows binding of TBP and RNAP?
The fact that DNA of an active promoter is usually free of nucleosomes
Is nucleosome localization stagnant or dynamic?
Dynamic - the nucleosome ‘moves’
What can change the location and density of nucleosomes?
The chromatin remodeling process
What accomplishes the chromatin remodeling?
The function of histone modifying enzymes and chromatin remodeling proteins
What does linker histone (H1) do?
Inhibits transcription
How does H1 (linker histone) repress transcription?
By occupying the position of DNA to prevent binding of transcription activators and RNAP
How did we find out an active promoter is free of nucleosomes?
Use restriction enzymes to cut a circular viral minichromosome within the promoter and enhancer region and have another enzyme cut a site opposite in another minichromosome. Look under microscope and see the cut within P/E has nuclesome free DNA at the ends, but the cut opposite P/E has has nucleosome free DNA in the center. Therefore, P/E is free of nucleosomes
What does chromatin remodeling (chromosome remodeling) do and what is it dependent on?
Changes the chromatin architecture and it dependent on ATP
What does it mean to remodel chromatin?
Change the position, density, or architecture of core nucleosomes
What causes the chromatin remodeling?
Histone replacement, histone protein modifications, nucleosome unwrapping and migration
What are some chromatin remodeling proteins which consume ATP?
SWI/SNF, SWR, ISWI, INO80, NuRD
What effect can chromatin remodeling have on transcription?
It can activate or repress it.
Summary of chromatin remodeling process
chromatin remodeling complexes hydrolyze ATP to change the nucleosome position or density
Mechanisms which facilitate chromatin remodeling (4)
- Mobilize nucleosome position (sliding)
- Dissociation of DNA-histone contact (unwrapping)
- Remove core histones (histone eviction)
Replace common core histones; e.g. H2A –> H2AZ isoform (histone variant exchange)
How does chromatin remodeling by core histone exchange work?
A remodeler protein repositions nucleosome to allow for a nucleosome free region and another remodeler protein faciliates replacement of core histone in the flanking nucleosomes of the nucleosome free region
Why would you want to remodel chromatin?
Nucleosomes tightly packed by common core histones have low transcription acitivity, but with a stable, nucleosome-free region, active transcription can occur
How do histone modifications facilitate chromatin remodeling?
They can loosen histone attachment to DNA, making it easier for the remodeling proteins
What’s an example of a histone modification and what facilitates it?
Histone tail acetylation by histone acetyltransferase
What is a histone tail and where is it located?
N-terminal 11-36 amino acids of core histones; not buried inside the nucleosome
How can the same residue of a histone be multiply modified?
By different chemical reactions under different conditions
What will the same chemical modification on different residues of a histone do?
Possibly have different effects on transcription
What are histone codes?
The combined pattern of histone modifications
What does the histone code H3R2mK4mK9acK14ac mean?
Histone H3 with Arg2 methylated, Lys4 methylated, Lys 9 acetylated, and Lys 14 acetylated
When is histone code important to transcription?
In vivo
What are the four major histone modifications?
Acetylation, methylation, phosphorylation, and ubiquination
What’s an acetyl group?
-C=OCH3
What’s a methyl group?
CH3
Whats a phosphoryl group?
-OPO3 2-
Whats a ubiquitin group?
A squiggly clump
What is acetylation?
Addition of an acetyl group to LYSINE residues
How does acetylation affect histones?
Have fewer positive charges so interact less strongly with DNA
How does acetylation differ in euchromatin and heterochromatin?
In euchromatin it’s hyperacetylated (common) while in heterochromatin it’s hypoacetylated (rare)
Where does methylation occur?
Often the same Lys residue as acetylation and in Arg
How do methylation and acetylation interact?
Often compete in Lys residues
What does phosphorylation do?
Introduces negative charges
What does poly-ubiquitination do?
Leads to degradation of ubiquintinated proteins
What is mono-ubiquitination?
Adding 1 Ubq; is not a degradation signal and is part of histone code
What acetylates histones
Histone acetylatransferase (HAT)
How does histone acetyltransferase (HAT) work?
Transfers acetyl groups from donor acetyl-CoA to lysine (not arginine) residues
What deacetylates histones?
Histone deacetylase (HDAC)
What effect does lysine acetylation have on histone?
Neutralzies positive charges of histone which tends to reduce interactions between neighboring nucleosomes, which decreases overall chromatin condensation so it usually activates transcription
How is HAT related to transcription?
Transcription co-activators often recruit HAT to activate transcription
How is HDAC related to transcription?
Transcription co-repressors often recruit HDAC to repress transcription
What proteins assist HAT recruiting?
When Max binds Myc, the dimer recruits HAT to acetylate histones and activate transcription
What proteins assist HDAC recruiting?
When Max binds to Mad, the dimer recruits HDAC to deacetylate histones which suppresses transcription
What does Mad1 dow hen it interacts with the Sin3-HDAC2 compelex?
It represses transcription
What happens when Mac binds to Mad1
the Max-Mad1 dimer recruits HDAC2 via the Sin3 bridge and HDAC2 deacetylates histones around the promoter region, resulting in transcription inhibition
Examples of transcriptional co-repressors
histone deacetylases
Examples of transcriptional co-activators:
histone acetyltransferases
What happens with hypoacetylated chromosomes?
Repressed chromatin and no transcription
What happens with abundant histone acetylation?
Active chromatin and transcription occurs
What happens to the thryoid receptor without thyroid hormone?
The thyroid receptor bind co-repressor NcoR to recruit Sin3, which binds HDAC to deacetylate histone and repress transcription
What happens to the thyroid receptor in the presence of thyroid hormone?
The thyroid receptor binds to a co-activator which is a HAT complex what acetylates histone to activate transcription
What’s an example of a repressor converted to an activator?
Thyroid receptor is a repressor without thyroid hormone, but with thyroid hormone, the histones are acetylated and transcription is activates
How can you use ChIP to identify chromatin DNA bound by a protein in vivo?
Fix chromatins, cut with ultrasonication or nuclease, immunoprecipitate with antibodies to transcription factor or modified histones; PCR the DNA from the promoter – the DNA of chromatin fragments bound by the protein will produce the PCR product
How did we find out histone acetylation occurs before transcription?
Infected cells with a virus and isolated chromatin at different stages of infection then ChIP against TBP or acetylated histone and PCR – see the acetylation occurred after viral infection but before TBP binding and mRNA expression
How does histone acetylation affect DNA-TBP interaction?
Shows that the transcription repressor Mad/Max co-repressor recruits HDAC to deactylate histone and suppress transcription but TBP recruits HAT to acetylate the histones at the promoter – ultimate level of transcription is dependent of balance between HDAC and HAT
Where can a histone be methylated?
At lysine and arginine residues
What do methyltransferases (HMT) do?
Transfer 1-3 methyl groups from donor SAM to lys or arg residues of histone tails
What effect does methylation have on charges of lysine and arginine and whats the resulting effect?
Doesn’t reduce the positive charges so does not reduce nuclesome condensation
What effect does hsistone methylation often have in general?
Chromatin condensation and transcription inactivation (but not always!)
Example of histone methylation repressing transcription:
H3K9 is methylated and binds to a chromosome remodeling protein which recruits HMT which methylates the next nucleosome at the same spot which eventually results in chromatin condensation (heterochromatin) which can cause gene silencing unless gene is insulated
What is the prokaryotic holoenzyme of RNAP
alpha, beta, beta’, w, sigma factor
What is the closed complex in prokaryotic transcription?
Holoenzyme + promoter DNA
What is the recognition seq and location for promoters in prokaryotic transcription?
TATA box at -10 position
Where are DNA regulatory elements located for prokaryotic transcription?
Usually close to or within the promoter and transcribed region
What regulates prokaryotic transcription?
A few transcription factors, changing of sigma factors, or RNAP for phages
What don’t prokaryotes have that eukaroyotes do in regards to transcription?
Histones, nucleosomes, complex chromatin structures
Components of eukaryotic transcription?
Three RNAP
Which RNAP transcribes mRNAs in eukaroyotes?
RNAPII
What is the holoenzyme of RNAPII in prokaryotes compared to eukaroyotes?
More subunits and requires TFIIs
What is the preinitiation complex in eukaryotic transcription?
Holoenzyme + TFIID + promoter DNA
Where are DNA regulatory elements located for eukaryotic transcription?
May be close to, within, or far away from promoter or transcribed region?
What regulates eukaryotic transcription?
Many transcription factors, co-activators, co-repressors, nucleosome position/density, histone codes, and aspects of chromatin structure
