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just know them. ALL OF THEM.
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repressors and activators that bind to DNA regulate via two different mechanisms:
- 1) they act with other proteins to modulate chromatin structure (inhibiting or stimulating ability of Tx factors to bind to promoters)
- 2) interact with mediator of Tx complex (mediator); this complex binds to pol II and regulates assembly of preinitiation Tx complexes
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nucleosome
consists of ~147 bp of DNA wrapped around 8 disk-like histones (2 of each of the 4 different types); is the basic structural unit of chromatin [just the associated DNA/protein complex]
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histone tails
-the N-terminal region of each histone (the C-term region of histones H2A and H2B) that extend from the nucleosome and can be (reversibly) modified to change the condensation of chromatin/it's accessibility
-the tails can be accessed by anything in the cytoplasm (aren't they in the nucleus?)
-the histone core is passive; TAILS are what change the configuration of chromatin
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heterochromatin v. euchromatin
- hetero = inactive/tightly wound; less accessible
- euchrom = actively Tx-ed, wound more loosely
-regions of chromosomes near the CENTROMERES and TELOMERES tend to be organized into heterochromatin (to preserve, protect and to prevent degredation)
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4 different types of histones:
- -two of each form the octamer that DNA is wound around; DNA + histones = nucleosomes
- -in some cells (like sperm), different proteins can replace histones and serve as anchors/condensors for DNA
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ubiquitization
the addition of a small regulatory protein (ubiquitin) that directs protein recycling/degredation
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HDAC
histone de-acetylase; enzymes that REMOVE acetyl groups from an acetyl-lysine amino acid on a HISTONE; is the opposite of HAT (histone acetyltransferase
-associated with more tightly wound (difficult to Tx) chromatin/DNA
-ex. yeast SIR2, and kind of RPD3
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HAT
histone acetyltransferase; enzymes that ADD an acetyl group to lysine amino acids on HISTONE tail; linked to Tx activation/euchromatin (loosely wound)
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HMT
histone methyltransferase; enzymes that catalyze the transfer of 1-3 methyl groups to lysine/arginine residues of HISTONES
-associated with deactivated/tightly wound (hetero)chromatin
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Dnmt
DNA methyltransferase; catalyzes the transfer of a methyl group to DNA; ADDS methyl groups TO DNA (as opposed to histone tails) so still promotes a less tightly wound conformation
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acetylation of lysines _________ their _______ charge & eliminates their interaction with DNA phosphate groups. This _______ chromatin condensation.
acetylation of lysines neutralizes their POSITIVE (+) charge & eliminates their interaction with DNA phosphate groups. This reduces chromatin condensation.
- -lysine residues have +positive+ side chains
- -DNA is negative b/c of the sugar/-phosphate- backbone
- -acetylation = the same as neutralizing lysine's positive charge (so it's NEGATIVE)
- -chromatin spreads out
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methylation functions to promote chromatin condensation....how?
- (talk about HP1 and methyl transferase)
methylation of specific lysine residues (parTICularly K9) in histone H3's tail attracts HP1 (heterochromatin protein 1)
-HP1 binds to histone methyltransferase; which in turn methylates H3 K9's on neighboring nucleosomes
-the methylation recruits more HP1 to bind to methylated K9's and the cycle repeats
-it is the associations between HP1's themselves that cause the tightly wound conformation of heterochromatin
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Polycomb proteins
essential for repression/maintaining repression of genes; recruited by a repressor; acts by methylating histone tails and packaging chromatin into inaccessible conformation; this complex STAYS bound even if the repressor is lost or unbinds; it extinguishes a gene forever
- -composed of 2 subunits:
- PRC1 & PRC2 that function to maintain the methylation and......
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Trithorax complex
includes a histone methyltransferase that methylates Histone H3 K4 (lysine 4); this modification is thought to create a binding site for histone ACETYLASE and chromatin remodeling complexes that PROMOTE Tx and PREVENT methylation at H3 K9 and K27
·trithorax opens forever: doesn’t mean always Tx-ed; just always available
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methylation at lysine 9 & 27 contribute to chromatin condensation via 2 different mechanisms: discuss
1) methylation at H3 K9 promotes condensation through the HP1/methyl transferase system
2) methylation at H3 K27 promotes (permanent) condensation through the polycomb complex (specifically PRC1) system
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epigenetic inheritance
type of inheritance through through modifications of chromatin structure RATHER THAN modification of DNA sequence
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mechanisms of repression that are more temporal than polycomb complex:
·competition: when a repressor binds to the same DNA site that the activator needs to bind to; essentially 'kicks' it out/prevents it from binding
·quenching (short-range): when the repressor binds next to activator and creates some sort of conformational change in it that prevents activator from doing its job; this method is only effective for local activators (or those right next to the repressor); far away activators can still function
- ·long-range: repressor talks to general transcription
- machinery and shuts down the gene as a unit
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repressors often work by binding to:
-co-repressors (ex: groucho, CtBP); these in turn recruit HDAC (histone de-acetylation) to remove acetyl groups and promote condensation
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RPD3 functions in a co-repressor complex
yeast protein (made from RPD3 gene) that is required for normal repression of a few yeast genes; also has histon deacetylase activity
- -needs to other proteins to deacetylase histones:
- 1) UME6: repressor that binds to URS1 (specific upstream regulatory sequence)
- 2) SIN3 part of a multiprotein co-repressor complex that also contains RPD3
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activators function by binding multisubunit co-activator complexes:
- SAGA complex: co-activator complex; most pertinant subunit = GCN5, which is has histone ACETYLase activity
- GCN4: actual activator
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nucleosomes at promoter regions of practially all ACTIVATED genes are:
hyperacetylated (spread out so Tx factors can get to the promoter)
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there are high levels of H3 K4 near:
active basal promoters
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acetyl groups on histone lysines (K's) turn over _______, whereas methyl groups are much more ______
acetyl groups on histone lysines (K's) turn over rapidly, whereas methyl groups are much more stable
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Chip-Sequencing (ChIP-Seq)
used to analyze transcription factor interactions with DNA-binding sites; it combines chromatin immunoprecipitation (ChIP) with DNA sequencing (using PCR)
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Process of Chip-Sequencing
- 1) DNA-binding proteins (Tx factors or histones in living cells) can be cross-linked to the DNA that they are binding
- 2) by using an antibody specific to a DNA binding protein, one can immunoprecipitate the
- protein–DNA complex after you lyse a cell
- 3) (crosslinking happens using formaldehyde to the cells)
- 4) after crosslinking, cells are lysed & DNA is broken into shorter (0.2–1.0 kb) fragments via sonication
- 5) immunoprecipitation is then done resulting in the purification of protein–DNA complexes
- 6) purified complexes are heated to reverse formaldehyde cross-linking of
- the protein and DNA, seperating DNA from the
- proteins
- 7) the identity/quantity of DNA fragments isolated can be determined by PCR
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