Meeting 16

just know them. ALL OF THEM.

• 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

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]

-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

• 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)

• H2A
• H2B
• H3
• H4

• -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

the addition of a small regulatory protein (ubiquitin) that directs protein recycling/degredation

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

histone acetyltransferase; enzymes that ADD an acetyl group to lysine amino acids on HISTONE tail; linked to Tx activation/euchromatin (loosely wound)

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

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

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

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

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......

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

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

type of inheritance through through modifications of chromatin structure RATHER THAN modification of DNA sequence

·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

-co-repressors (ex: groucho, CtBP); these in turn recruit HDAC (histone de-acetylation) to remove acetyl groups and promote condensation

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
• 

• SAGA complex: co-activator complex; most pertinant subunit = GCN5, which is has histone ACETYLase activity
• GCN4: actual activator

hyperacetylated (spread out so Tx factors can get to the promoter)

active basal promoters

acetyl groups on histone lysines (K's) turn over rapidly, whereas methyl groups are much more stable

used to analyze transcription factor interactions with DNA-binding sites; it combines chromatin immunoprecipitation (ChIP) with DNA sequencing (using PCR)

• 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