Histone Modification Flashcards
Describe classic epigenetic phenomena studied in drosophila
In Flies = not due to dna meth but still have interesting effects
Position effect variegation
Antennapedia
Describe position effect variegation
Eye colour - white or red
What determines = position effect variegation
Epigenetic phenomena
Heritable gene silencing by heterochromatin.
Makes patterns = heterochromatin forms over one of gens responsible for eye colour
Describe antennapedia
Loss of silencing - failure of regulation of hoax genes at wrong time in dev
If turn on at wrong stage dev = legs frowning out of head where antenna should be
What are classic epipgenetic phenomena in drosophila due to
All due to histone mods
NO DNA METH in flies
What is nucleosome
Fundamental unit of chromatin structure
DNA, histone, octamer with core histone = H2A, H3, H4, H2B, 2 copies of each
Defined structure for most of histone protein
Histone tails
Describe histone tails
Where most of histone mods occurs
Some parts do not have as much structure = tail domains
N terminal tails
Name and describe some types of post translational histone mods
Diversity
H2A = ONE OF COre histones, tail at n terminus for all, 100-1200 aas long - small ish (all)
Post translational mods at many sites = some can have many mods, chemical complexity
H2B, H3, H4
Many types co occur = clustering in groups, seemingly functionally related
What are markers for chromatin state
Histone mods
Describe heterochromatin markers
Not transcribed, inert
Hypoacetylated histones
DNA meth
H3k9 meth
H3k27 meth
More dense and compacted
Describe euchromatin makers
Trasncriptionally active
Hyperacetylated histones
H3k4 meth
H3k36 meth
H3k79 meth
Less electron dense than heterochromatin
Describe acetylation of lysine side chains - histone mods
Lysine residues in histones
Correlation between histone mods and activate states which are present near gene
Describe histone acetylation
Aliphatic chain, + charge amino group
Acetyl co a = donor molecule
HAT = histone acetyl transferase
Histones rich in arginine and lysine (+) interact with phosphate back bone (negative charge)= helps form nucleosome complex
Acetyl covalently attaches to terminal amino group - no longer + charge
So changes interaction with dna = favours less tight interaction with histones and dna = more permissible for things to come and interact with dna
Mostly on histone tables
REVERSIBLE
What do hdac do
Turn genes off
What does acetyl group do - histone acetylation
Neutralizes positive charge, = abrogates interaction with dna
Charge based mechanism
What is corelated with transcriptional activity and dynamic chromatin structure
Histone acetylation
What are hats and hdacs involved in
Hat enzymes = transcriptional activation
Hdac = repression (including heterochromatin)
What else does acetylation do
Creates a binding site for other chromosomal proteins
Charge neutral but creates dining site
Describe what can now bind after acetylation
Acetylated lysine fits into biding pocket
B
Transcription activator proteins contain domains that can bind = bromodomains - one of types of protein domains that read histone mods, Found in transcription activator complexes= READER DOMAINS
What does acetylation serve to do
Recruit other important regulators to chromatin
What is histone lysine methylation
Another mod
Not charge based mechanism but still creates binding sites for other proteins
Replaced proton with methyl in amino group = can be mono di or tri methylated
What does lysine methylation
Usually catalyze by enzymes containing a set domain = catalytic domain
NO CHANGE IN LYSINE + CHARGE
Describe histone tails specifically
Chromosomal signalling platforms
Binding site creation for other proteins = main mechanism
When modified = signal to proteins to bind - affects transcription
Dictates effect on transcription
Can be acetylated, phosphorylated, methylated
Reader domains bind
Name reader domains that bind to histone tails
Chromodomain - tri methylated lysine
Bromodomain - acteylated lsyine
How are histone lysines demethylated
Oxidation of methyllysine = common theme with dna meth
2 kinds of enzymes = LSD and JmJc enzymes
Diff mechanisms
What can oxidize me1, me2
LSD EnZYMES= di and mono
What can oxidized all 3 methyl lysine states
JMJC
Describe what characterizes heterochromatin
2 histone methylation events
How heritable gene expression states created and maintained and passed on
What type of lysine methylation is for constitutive heterochromatin
H3K9
Histone mod on histone 3, lysine 9
Linked to constitutive heterochromatin
What type of lysine methylation is for facultative heterochromatin
H3K27 methylation - lsyine diff
(Sometimes also H3K9me - dual role, but complicated)
Diff roles = diff outcomes
What are essential for heterochromatin function
Histone methyl transferases
Describe histone methyl transferases
2 sets of enzymes = each conserved to one another through evolution
Evolved enzymes for specific sites
On same protein, lots of specificity
Name and describe the families of histone methyl transferases
Ones that bind to K9= suv39h1, suv39h2, setdb1, G9a, glp
Ones that binds to k27 = ezh1,ezh2
Why do we need the histone methyl transferases a
Histones v evolutionarily conserved
Essentially the same in yeast as in humans, need diff family members to methylate site
What do all histone methyl transferases have
Set domain= catalyses histone methylation
What are binding sites for specific heterochromatin proteins
Modified histones
Same specificity applies to readers
Describe histone readers - specifically
Families of protein domains recognize sites after modifications
Describe specifically the readers that bind to specific sites
Hp1, cbx1,3,5 = binds to k9, (heterochromatin protein 1, in flies)
Polycomb, cbx2,4,5,7,8 = binds to k27 (also fly protein)
Has homologs in humans
What do hp1 and polycomb proteins share
Hp1 and poly comb proteins all share a chromodomain, a protein domain that binds to methylated lysine
How do we map histone modifications and chromatin proteins on chromosomes
To determine histone mods = do chip, chromatin immune precipitation
Describe regular immunoprecipitation
Recognizes the one protein
Use antibody to locate protein of interest
Describe chip
For chromatin = antibodies can recognize specifically modified histones, also can do it against any protein of interest that you think is bound to chromatin
Make mixture of proteins in a way that breaks up chromatin but still preserves interactions between histones and dna = use cross linking technique and use antibody = fish out specific histone while still bound to dna
Do ip = get rid of proteins and look at dna, usually comes bound to dna and nucleosome
= can do pcr, q pcr, microarray, sequencing
Genome wide pic of where modified histone bound in genome
What can heterochromatin do
Cover large contiguous chromosomal domains - genome wide
Can use chip seq
Can map peaks in genome for diff mods
Describe role of h3k9me in differentiation of early embryo
Use chip = can see how changes during dev
Ex = look at where mod is in early vs later dev and how it changes (pattern)
Sees role of heterochromatin and how it regulates dev during differentiation process
Gastrulation stage = get cell form mouse embryos and see more cell types = endoderm, mesoderm, neural ectoderm
= analyze with chip
What is result of h3k9me chip seq in early embryo
Shows locus specific effects during differentiation
Compared early to later stage = more or less methylation in certain regions
Changes in facultative heterochromatin = saw it forming and being removed
Comparison of h3k9me3 patterns in cell populations isolated from diff developmental stages
Differentiation events associated with gain and loss of h3k9me3 at diff genes
describe exp with endoderm
Elimination of h3k9me = less heterochromatin visible
Conditional KO removing setdb1, suvar39h1, suvar39h2 specifically in undifferentiated endoderm = E8.25 (specific time)
= KO what catalyses H3K9me (removed Almost all of it)
What is conclusion of endoderm KO exp
Defects in hepatocyte differenattfion although many animals survive to term= Looks specifically at liver = didn’t differentiate properly
Heaptocytes (liver cells) express many non linage genes = genes that shouldn’t be on
CONCLUSion = H3K9me dependent facultative heterochromatin is essential for proper differentiation
What binds to h3k9me and mediates transcriptional repression
HP1
= need reader molecule to help create modification state
Has chromodomain = recognizes H3K9me
Mediates transcription repression and heterochromatin formation
HP1 binds and does something = biophysical transformation = creates more compact state= mechanism of repression related to compaction of chromatin structure mediates by unique physical properties of chromatin bound to HP1
What do biophysical properties of hp1 help create
Heterochromatin foci in cells
Ex = hp1 proteins in test tube, exogenous setting = forms weird globules = unusual self association property
Also ex = hp1 visualized in cell= not just spread evenly - forms big foci, has some property to create condensed regions of chroms
What does polycomb silencing require
H3k27 methylation and is a specialized facultative heterochromatin system
Helps keep developmentally regulated genes in off state when need to be off
Describe polycomb silencing
Carried out by a class of developmental regulators = polycomb group or PcG proteins = originally discovered in drosophila, regulates hox genes in flies and mammalian cells, defects if not work
Describe loss of PcG silencing in flies
Developmental transformation - where cells destined for antennae turn into legs
Describe loss of polycomb proteins in mammals
wide range of dveleopamntal phenotypes=gastrulation defects, transformation of axial Skelton, hematopoietic and neurological defects (similar in orgin)
In mouse = bad defects, hematopoietic, gastrulation
What do conditional KOs show about polycomb silencing
Evidence of loss of terminal differentiation in adult tissue = required continuous during dev to keep cell differentiation wired correctly
What is polycomb silencing = what kind of system
Functionally distinct from h3K9me and dna meth = PcG is a dedicated facultative heterochromatin system = own system
Describe polycomb proteins generally
2 major functional units = coresspond to biochemical complexes = prc1, prc2
What does prc stand for
Polycomb repressive complex
Describe prc2
Has h3k27 methyl transferase, had aebp2, jarid2, = binds to ub histone
H3k37 methyl transferases complex
Catalytic subunit ezh1/2 (mostly ezh2), aebp2 and jarid2 subunits that bind to ubiquitylated histone H2A
Describe prc 1
Binds to h3k27, can mono ub histone H2A
Contains cbx chromodomain proteins that bind to H3k27me, also contains rnf2 (also called ring1a, ring1b) histone H2A ubiquitylation enzyme
What happens - describe polycomb prc 1 and 2
Prc 2 = put on meth, prc 1 = bind to meth (chromodomain)
Prc1 = put mono ub, prc 2= aebp2, Jarid2 binds ub histone
H2Ak119ub1 and h3k27me both localize to polycomb repressed genes and reinforce one another
= feed forward loop= connects complexes of both these proteins- drive each other, recruited by to chromatin
Where is repression function - which prc
Prc1
Describe what creates repression by prc1 - mouse exp
Compaction of nucleosomal arrays by mouse prc1 proteins
Self association compartments
Strings of nucleosomes = chains of histones
Prc1 creates big clump create compact nucleosomal Structure
Atomic force microscopy images of nucleoside arrays
Biophysical mechanism seems related to that use by hp1 proteins - inherent ability t form some kind of self association compartment
What is required for oligodendrocyte differentiation
Polycomb system
What are oligodendrocytes
Cns cells
Form myelin sheath around axons
Describe conditional KO involving oligo dendrocytes - exp
Eed (part of prc2) ko in oligodendrocyte precursors = prevent myelination= no sheath
Describe conditional KO involving oligo dendrocytes - results
No myelin sheath = due to defect in oligodendrocyte precursor differentiation - opus actually switch and end up as astrocytes= cellular differentiation problem
Why does cell fate switch in oligodendrocyte precursors
Traced back to specific effect on gene expression cell fate switch is due to failure or reps wnt and bmp signalling pathways in precursors = activate signalling pathway when should not = differentiate into dif cells = increases expression of wrong genes, associated with inappropriate signalling pathways
Transcriptional targets of these signalling pathways needs to be repressed by polycomb system in precursors - need repression by prc2
How to dna/h3k9/h3k27 methyl transferases get to their target organs - gen
Do not want them all over genome -only specific parts
Must find specific genes in genome
No actual answers but think of 2 general ways
How to dna/h3k9/h3k27 methyl transferases get to their target organs -interactions
Interaction with other chromatin factors - like histones or specifically modified histones
In some cases = interaction with site specific dna binding proteins like tfs, recognize specific sequence
How to dna/h3k9/h3k27 methyl transferases get to their target organs -polycomb
Polycomb: interaction with specific genomic dna sequences = unmethylated CpG islands = some connections
Polycomb: interaction with non coding rna = transcribed from diff regions genome and have role in targeting prc 1 and 2
Describe cross talk between histone methylation and dna methylation - gen
Connections = occurring in many cases and specific places
Strong relationship between dna meth and meth k9
2 domains - can bind to both
Describe cross talk between histone methylation and dna methylation - specific binding
Uhrf1 tandem Tudor domain (ttd) binds to h3k9me (epigenetic reader proteins for hsitone mods)
Uhrf1 SRA domain binds methyl CpG - binds methylated dna
Uhrf1 and dnmt1 interact = stronger link since interact
Creates self reinforcing loop linking dna methylation to h3k9me - in some places of genome, act together and reinforce creation of certain kinds of heterochromatin
Describe cross talk between histone methylation and dna methylation - methyl dna binding proteins
Mecp1, mecp2, mbd family all form complexes with hdac enzymes = turn genes off, deacetylases found to interact with proteins that interact with methyl dna
Link dna meth to an action on histone that also helps turn genes off
Linked histone deacteylation to dna meth
Describe cross talk between histone methylation and dna methylation - other dna methyl transferases
Have chromatin targeting domains = dnmt3a, to get to specific places in genome
What do histone tails have - cross talk
Histone tails= determine where these factors localize - ADD DOMAIN = histone tail binding domain
Describe ADD domain
Can bind to unmodified tail of histone h2 but if methyla h3k4 = blocks add domain
What does dnmt3 have - cross talk
Dnmt3 add domain binds to h3 tail only when h3k4 (euhcromatin mod) is unmethylated, not modified
What is h3k4 me associated with
Promoter regions of transcribed genes = CGIs
Happens a lot in promoter regions aossciated with CpG islands
Overal - what does add domain + cross talk do
Prevents dnmt3 from methylating CGIs near actively transcribed genes = contributes to the unmethylated state of promoter CGIs
Mechanisms to exclude dnmt3 from binding =blocks dna methylation from being introduced in these regions
