Chromatin Structure 1 + 2

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  • Created by: Sarah
  • Created on: 17-05-19 12:58
where is heterochromatin transcriptionally silent chromatin found in the nucleus?
in the periphery- associated with nuclear M
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what are interphase nuclei made up of?
discrete chromosome terroriteis subdivided into activae and inactive chromatin
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what is the key gene for sex combs formaiton?
sex combed reduced
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towards the centre of the nucleus what are domains associated with?
transcriptionally active chromatin so associated with TFs like RNA polymerase 2
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what do RNA polymerase 2 seems to be localised to
distinct chromosomes- active ones
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what are the bundles in the nucleus known as?
topologically associated domains- are inactive and active domains discrete entities
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what has allowed us to deal with the huge volumes of data rich information?
computational tools and software
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what does mutant suvar do?
are all red eye
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what can bicohemical tools tell us?
protein and nucleic acid composition and the distrubition of chromatin mods (acetylation and methylation)
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what happens when you remove Evar?
normal function is to promote transcription of white allele (red phenotype)-
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what is suvar3-9?
a histone H3K9 methytrasnferase- puts methyl gorups on lysine 9
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what did genetic techniques allow?
to see impact of specific molecular changes on organism, physiology, development and behaviour
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how did they use to tell when CpG were methylated?
using REs that selectively recognise unmethylated or methylated CpG rich sequences
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how did they use to look at methylation originally?
by focusing on a specific gene and specific nucleotide pair
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now what are we able to do?
analyse modification patterns across the entire genome or large chunks of chromosome
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how do you look at these widespread DNA changes?
antibodies specific for chromatin modifications eg acetylation or methylation or proteins that are readers+writers
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what does meDIP stand for?
methylated DNA immunoprecipitation
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how does meDIP work?
anti-5-methylcytosine antibody to pull down and select out genomie fragments that contain DNA mods (eg CG methylated ones) allows you to identify sequences associated with modifications
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how do you identify which genes they are?
by sequencing the genes
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what is bisulfite sequencing analysis of genomic DNA used for?
to map and quantify methylated CpG sites so basically where they are
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what does MspI cut?
both methylated and unmethylated DNA
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what does HpaII cut?
methylated CGsq
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how do you uese REs be used to find DNA methylation?
1) digest genomic DNA with MspI or HpaII 2) electrophose in agarose gel 3) transfer DNA to nylon M by southern blotting 4) hybridise nylon m to radiolabelled DNA probe with sequence matching gene of interest
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what does it tell you?
if its sensitive to methylation then HpaII doesn't cut methylated sequences so DNA fragments will be much bigger in the HpaII digest then the MspI digest
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what is the DNA probe labelled with
radioactivelly labelled P32
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what is one of the flaws of the immunoprecipitation technique?
limited by how good the antibodies are
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what is the other technique
biochemical technique using REs
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what does HpaII sensitivity allow you to do?
map the distribution of individual methylated CpGs across a region of DNA (providing you have probes so know the gene you're interested in)
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what's an example fo HpaII sensitivity in use?
tissue transglutaminse promoter- has differential sensitivity to HpaII but not MspI
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what did they do to see the transglutimiase promoter methylation?
studied HeLa cells- digested with Hpaii, mspI and HpaII digest gave a mcuh longer fragment telling us CCGG is methylated here so can't be cut
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what happened when they treated the HeLa cells with 5a cytosine = inhibitor of DNA methytransferase?
no methylation so HpaII can cut and get smaller fragments produced = shows methyltransferase activity is inhibited
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what did this approach allow you to do?
analyse changes in specific methylation sites- so changes in the abundance of methylation in specific locations
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downside of it?
you need a probe
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what technique is used for genome wide analysis of DNA methylation?
meDIP sequencing
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how can meDIP sequencing be used for genome wide DNA methylation changes?
take HeLa cells -> extract DNA -> sonication to fragment DNA -> incubate fragments with antibodies for methylated CGs, pull down, align it to a reference genome, can make a map of where all methylation is but have primers to detect which seqs
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what is the technicality with the sequencing?
sequence them and count the number of times you sequence each fragmenr (Equal size + length fragments) - if its being sequenced a lot in the pulldown, its a very common sequence read-map methylation across the gene based on num of times u seq region
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the more times a sequence is in the pulldown then what?
the more methylation there is of that locus in that sample
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why can we compare methylation patterns across a gene or chromsomal region of interest?
got the mapping bioinformatic tools to assign sequences to particular locations in the genome
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what did they show for methylation in the cancer line chromosomal region being studied for methylation?
there's flcutating levels of methylation across the region of the genome for the red+green tissue. 2 differentcells fibroblasts and colon cancer have differential methylation+ hypomethylation in cancer cells active but silent in fibroblasts
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how can you extend meDIP to very large regions or entire chromosomes?
by aligning methylation profiles and determining methylation profiles across a whole genome
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where is NFKB expressed? what does it do?
in WBCs, activates T cell/ B cells makes them secrete secrete antibodies or produce T cell receptors
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what did the DNA methylation done using meDIP-seq show?
in memory T cells and the naive T cells there's no methylation across the NFKB gene all other tissues have high peaks (liver, foetal brain, breast) which is what you expect
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what did this study show?
you can get predictive power from your methylation analysis across T lymphocyte lineage have high NFKB expression
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what's another way of studying methylation?
bisulfite sequencing analysis
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how does bisulfite sequencing work?
sodium bisulfite will deaminate cytosines but won't deamintae them if they're methylated
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what does the deamination of cytosine turn it into?
cytosine deaminated into a uracil
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what happens if you gae a methylated CpG and treat with bisulifite?
nothing as the methylation prevetns bisulfite deaminating it to uracil
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how do you determine what happens to umethylated Cs?
will be sensitive to bisulfite and turned into uracil, analyse sequence PCR amplification, after multiple PCR UA -> AT
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what can you use bisulfite treatment for?
measure amounts of methylation in a tissue if it was unmethylated you'll have no CpGs at the end but if it was methylated you'll have lots, do it aross genome see eac CG contains methylated or unmethylated
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how do you find the % methylartion of each CpG pair in the DNA sample?
calculate the % reads that are at each CpG site, after alignment to a reference genome for each DNA sample
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what did they show with CpG DNA methylation plots across the hoxD locus in ZF?
CpGs are demethylated on maternal chromosomes as ZGA occurs in pluripotent blastomeres of sphere stage embryos
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example of using MeDIP seq to look at DNA methylation changes over time?
wiping out methylaition at ZGA of the hoxd locus
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what kind of gene is hoxd?
homeotic gene
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when is ZGA around?
around 512 cell stage
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is methylation high or low across the hoxd cluster in sperm?
low
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is methylation high or low across the hoxd cluster in the egg?
high
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what happens after fertilisation?
methylation has risen to match the egg- both sperm and egg genomes are highly methylated across that region and methylation persits through the maternal epipegenome is transferred to the paternal epigenome
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what happens at sphere stage when zygotic gene trasncitpion starts (ZGA)?
methylation is wiped off and hod cluster is prepared for its developmental role
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how are genome wide distrubitions of histone modifications and proteins in chromatin investigated?
CHIP = chromatin immunoprecipitation
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what is CHIP?
use antibodies specific for histone modifications (eg antibody specific for histone 3 lysine 27 methylaiton) or histone writers/readers
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example of CHIP in the mouse?
changes in histone 3 lysine 4 methylation in the oocyte compared to meythl cytosine
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what did this show?
where methyl cytosine levels are high, H3K4 methylation is low and there's a low level of acetylation in the oocyte
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what did it show you about lysine 4 methylation and lysine 27 acetylation?
they go together- where you have lysine 4 methylation you also have lysine 27 acetylation
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why do we study drosophila salivary gland polytene chromosomes?
in metaphase you can isolate them readily, easily visualised down a microscope as they're huge
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what particularly is studied with drosophila salivary gland polytene chromsomes? what's it made up of?
heterochromatin- at the chromocenter is an accumulation of heterochromatin (transcriptionally silent) made up of 4 sex chromosome pairs and chrs 1, 2 and 3
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what was shown to decorate the chromocentre- the region of heterochromatin(using antibodies)
H3 lysine 9 methylation mark- this marks heterochromatin rich, but no euchromatin marker (H34me2)
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what do histone modifications sharply define? with what marks?
domains of heterochromatin = H3K9 euchromatin = H3K4 methylation
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what is found at the chromocenter in drosopholia polytene chromosomes?
hetercohromatin rich in H3K9me2 = transcritpionally silent mark
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how did they show these markers for euchromatin and heterochromatin?
CHIP chromatin immunoprecipitation using antiboides for them modifications and map at the nucelotide sequence the distribution of H3K9 methylation and H3K4 mehtylation
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what is position effect variegation?
where translocation of a transcriptionally active gene near to heterochromatin can transcriptionally silence it, an active gene if it moves the gene closer to heterochromatin
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example of position effect variegation?
drosophila ommatidia
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what does the white gene code for?
red eye colour (when KO causes white eyes thats why its called that)
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what happens if you take WT white and induce chromosomal inversion that relocate closer towards heterchromatic blocks in the chromocentre?
it gets transcriptionally silenced- (suvar is methylating all around there causing it to be shut down)
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what happens to the phenotype when white translocates near to heterochromatin?
get white spots in the red eye where that white gene has done that
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what can the eye pigmentation phenotpye caused by chromosomal white gene locus rearrangement be used to identify?
mutations that supress or enhance position effect variegation- screen for genes that regulate heterochromatin properites
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position effect variegation will usually do what to a gene?
transcriptionally silence it (stuck in heterochromatin)
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what would enhancers of position effect variegation do to the phenotype?
they would enhnace transcriptionally silencing of white gene (red eyes) so you'd get completely white eye
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what would supressors of variegation do?
they would prevent transcirptional silencing so eye would still be red
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what are nehancers and supressors known as?
suvars and evars
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what do suvars do?
prevent position effect variegation so they supress the heterochromatin and allow white to be expressed = supress transcirptional silencing
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what do evars do?
enhance variegation so enhance transcriptional silencing so supresses white gene more
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what does suvar3-9 do?
methltransferase, methylates H3K9 so compacts all the heterochromatin up so white genes not expressed
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what happens in the suvar3-9 mutant?
can't methylate heterochromatin so white becomes transcriptionally active and so you get all red eyes in the suvar mutant
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what defines the chromocenter?
H3K9 methylation marks
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how do you look at H3K9 in suvar mutants?
take WT or mutant suvar flies, run a WB and probe for methylated H3 lysine 9
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what do you find? what did this tell us?
WT has H3K9 methylation but suvar mutants have no H3K9 methylation = suvar is a methyransferase that amkes methyl makrs on H3K9
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what domain does Suvar need to make methyaltion marks?
SET domain
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what protien recognises Suvar H3K9 methylation marks?
heterochromatin protein 1
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what does HP1 do?
bind to methylated lysine 9s (H3K9) that are made by suvar3-9
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what experiment told us HP1 binds specifically to H3K9 made by suvar3-9?
agarose column on which peptides from histones H3 that contain methylated lysine 9 or lysine 4 then add radiolabelled HP1 protein and pour onto column- find HP1 binds to H3K9 methylated oclumn not lysine 4
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where are trimethylated or dimethylated lysine 9, HP1 and suvar localised?
all co localised to the same region of chromatin to each other
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what is excluded from the domains with suvar HP1 and H3K9?
H3K4 methylation- activation marks, RNA polymerase 2
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suvar HP1 and H3K9 emerge?
at the boundaries of the heterochromatic regions
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what were the oocytes abundant in?
oocytes are associated with RNA pol 2 and abundant in chromatin regions that is NOT heterchromatin
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whats the function of suvar 3-9 making methylation marks and HP1 binding to it?
HP1 dimerises, the dimers bring 2 nucleosomes in association with the HP1 dimer. Acts as a short bridge to pull those nucleosomes together- constatinaing everything down = open -> closed chromatin
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why is relevant that chromatin is closed?
it's inaccesible to RNA polymerase
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what is one thing we're trying to find out about the nucleus?
find out whether there's consistent spacing within the nucleus of a particular cell type , constent chromatin codnestation and whether it differs in cell typees- do domains op open vs closed chromatin vary according to cell identity?
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what are some of the incoming factors to open chromatin?
TF or enhancer bidning proteins so DNA seq specific transacitvators that come and find an enhancer/promoter and recruit RNA polymerase
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how are regions of open chromatin accesible to chromatin modifying enzymes identified?
DNAase I hypersensitivity mapping
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what is DNAase I?
nuclease- enzymes that cleave DNA sequences- endonucleases cleave within a seq
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what other nucelase is there?
microlol
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both of microlol and DNAase I are what?
sequence non-specific endonucleases- degrade DNA from isnide out
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what region will these nucelases work on?
regions of DNA that are free and accesible
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what can this methology be used for?
to identify regions of DNA in chromosomes that are accesible
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how does DNAase I hypersensitivity work?
take cells, permeabilise them, chemically isolate the nuclei, permeabilise nuclei so they take up DNAase I from the solution they're bathed in and DNAase will chop up DNA where it's accesible
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what would prevent DNAase I getting to the DNA?
closed chromatin- DNA regions that tightly bound to nucelosomes because histone octamers prevent it accessing the double helix
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what regions does DNAase I chop up?
DNA in the open chromatin
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what are the 2 ends of the pieces of DNA usually defined by?
Restriciton sites made by bamHI- a RE used to cut genomic DNA for making southern blots make big smear of fragments so you can then hybridise your probes
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how do you detect the DNA on the southern blot?
P32 labelled DNA probe
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what happens if you treat the cells with DNAase I before doing a BamHI digest?
if they have accesible sites within their nuclei then BAM H1 fragment won't exist any longer or most of the cells will have lost it as DNAase I will chew that fragment up in the middle make it smaller so BAM H1 fragent will no longer exist
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how will you see this when you hybridise your probes to detect that BAMH1 fragment?
when hybridise your probe to detect the BAMH1 fragment if they have accesible regions endonuclease would have chewed it away so BAMH1 fragments you detect will be smaller
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what did they see when they did a DNAase I hypersensitivity experimetn with erythrocytes and fibroblasts?
on the southern blot you see truncated BAMH1 as there's accesibility to the gene in the ertyhroblats but fibroblasts do not
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what is used as a paradigm for understanding structure and function within chromatin?
the human beta globin locus
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what was the beta globin gene an example of?
DNAase I hypersensitive regions in chromatin allowed the researchers to home in on an enhancer region that directs the developmental and tissue specific transcription of the linked genes
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which genes does the beta globlin locus express and when?
episolibin + gamma globlin (expr in embryogenesis) then they're shut off and beta/fetal globlin stays on, delta comes on and in the adult bone marrow you see beta major expressed
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how are these genes expressed in what fashion?
genes are expressed in a developmentally regulated tissue specific fashion sequentially
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where in the beta globlin locus is rich in these hypersensitive
20 kilobase region at the side of the cluster = locus control region. LCR is rich in DNAase I hypersensitive sites
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what experiment showed the hypersensitive site 4 (accessible to proteins)on the human beta globlin LCR?
added increasing amounts of DNAase I. Take nuclei from cells and peremabilise them gently, add in the DNAase I for a period then extract the genomic DN, digest it with BAMH1, run WB and hybirdise to probe that detects BAMH1 fragments
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what did they see with erythrocytes when they did this?
only in erythrocytes you get truncated BAMH1 fragments = DNAase I endonuclease has chewed it up so identifies the specific region of chromatin in the vincitiy of beta globin gene that is hypersensitive and acccesible
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what does the human beta globin LCR act as?
a super enhancer- has all the accesible sites (hypersensitive region)
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what does the LCR confer?
position-independent trangene copy number dependent transcriptional activity to multiple transcription units in transgenic mice
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what did they do with that hypersensitive sequence they found int he LCR?
took that sequence and made a trangenic animal with it in which that fragment that was hyperaccesible (open chromatin- enhancer region lots of TF BSs) link it up to b-globlin promoter and coding seq
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then what did they do with the transgenic animals?
did a northern blot (proteins) studying the level of expression of that transgene in different transgenic lines
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what did they find with the transgenic lines?
some have much more mRNA, some show almost no expression
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how do they make these transgenic animals?
adding in this exogenous structure into the genome that gets incoprorated randomly an din various copie numbers so different transgenic lines and these are relative amount of mRNA priduced in these constructs and number are closely related
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what did this show about the relationship?
there's a direct relationshop between number of copies and level of expression- its copy number dependent and position of intergration independent transcription bcos wherever thos structures are they're integrated randomly
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What does that mean?
wherever that transgene goes regulatory elements in it aren't affected by location of transgene- in eu/heterochromatin that is not typically expressed in erythocytes that reg element confers RBC specific+position indep transc to be expressed in RBCs
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why are LCR called super enhancers?
they act in a really powerful way on the seqs near them on the chromosome but in the beta globlin cluster they provide developmental regulation so stage specific + lineage specific expression to the coding sequences
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how does the LCR only regulate the beta globlin gene?
only regulates the beta globlin gene because there are boudnary elements that constrain the activity of this LCR keep its attention them genes
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how can microccal nuclease be used?
allowing you to see very tight array of nucleosomes by getting into junctions between each nucleosome - classic way of detecting heterochroamtin = using microccal nuclease
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whats different about microccal nuclease to DNAase I?
its smaller so can access DNA regions that DNAase I finds it harder to get to
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how did they show nucleosomes with microccol?
used a position effect variegation alle- 2 different transgenic flies- 1 insert into euchromatin so red eyes and another where insert in heterochromatin, isolate nuclei southern blot to detect white sequences on white gene
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what did they find?
when in euchromatic region see smear of small fragments- breaking down fragments. but heterochromatic region isnettion transgenic animals0 1 nucleosome length of DNA 146 bp of DNA each = shows individual nucleosomes with micrococcal
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what is ATAC seq for?
genome wide analysis of chromatin accesibility using an assay for transposase accesible chromatic-sequencing (ATAC seq)
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what will ATAC seq give us?
an idea about the total accesibility of chromatin across the genome and compare it for different cell types and match that up to histone acetylation or DNA methylation
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what do DNA transposons do? where do they come from?
transposons from bacterias move from one location to the other inserting themselves create a duplicated sequence, generate seq, carry it into locus and place either side of the transposable element that goes in
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what do these transposons do once they're in?
look for regions of accesible chromatin and insert and mark all regions of chromatin that are avaliable to them with short sequence tags so they can extract the total genomic DNA, sequence it and map tag locations- map genome for accesible chromatin
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chromatin accesible to transposase is what?
DNAase I hypersensitive (accessible chromatin) and enriched in transcriptioanlly active chromatinstone modifications
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how did ATAC sequencing show this?
human chr 19- peaks of chromatin accesibilty with DNAase hypersenitivity are associated with transcription start site- ATAC seq where transposon reads have been found are associated with this region map perfectly
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how do you get tags from transposons?
they cut and paste their bits into the bits of accesible chromatin they've attached to
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what did they tell about chromatin accesibility in early ZF embryogenesis?
chromatin accesibility is developmentally regulated- it increases at large genes numbers during ZGA
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what can you see about chromosome accesibility for genes as development continues past ZGA?
peaks of ATAC seq sensitivity accesibility gets stronger as development proceeds so intially not affected by ATAC but as ZGA begins accesibiliuty to external agents increases- opening up of genome, p much all genes have ATAC seq associated with them
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what accompanies this opening up of chromatin?
DNA demethylation, demethylation of paternal CPGs to maternal chromosomes as ZGA occurs in pluropotent blastomeres at the sphere stage
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what technique would allow us to know if DNA sequences are close together in the 3D nucleus and if these proximities change and what seqs are next to each other?
chromatin confomation capture
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what does chromatin conformation capture ask?
which sequence sexist in close proximity to each other?
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how does chromatin conformation capture work?
take chromatin from isolated nuclei, chemically crosslink DNA to protein (formaldehyde), add lots of nucleases, digest DNA
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What do you get left with?
doublets, short pairs of DNA sequences that are held together by a TF or histone protein but these doublets are regions of DNA were close togther in the nucleus before
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what do you do with that sequence info?
make a library of those seqs, can DNA ligase them make different ircular molecules, get rid of proteins, reverse crosslinks then sequence them see what u have
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why do you need a reference genome?
have to map every sequence back to a reference genome and ask which sequences is every sequence associated with so pairwise comparison of sequnce content
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how would you come up with a statistical model of what the 3D structure of that region looks like?
count the amounts of the different combinations of which seqs are interacting, seqs not in touch won't come back as seq elements within the same conjoined fragment as not close together
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where is the beta glonblin gene mainly active?
erythrocytes
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what model did they get for the beta globlin gene with chromatin conformation capture?
there's a loop in which the transcribed region- the beta major gene here, the episolobin gamma gene exist in a loop, at the neck of which lies the LCR- TF sit on the LCR and this stable neck of the loop sequentially contacts each gene in turn
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what supports this model?
frequency with which this fragment here in the middle of the LCR is contacting other seqs across this region- frequency changes as a function of position, erythoblasts+fibroblasts diff
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how does this frequency change?
peaks of high corsslinking freequency (so whenever that was sequences that was sequenced with it)- tells you that fragment is often in touch with this region
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what happens if you flip the analyses around and do a complementary experiment where you look at interactions this regions makes with the rest of the locus?
come out with the same result- most popular interactor is the region in the middle of the LCR- fibroblasts very little interaction compared to erythoblast interaction
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what did that data tell you?
there's a loop her ein which the LCR that contained hypersensitive sites including HS5 is ofern in touch with 3' HS1 (binds)
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what binds to the DNAase I hypersitive sites HS5 and 3' HS1 of the human beta globlin LCR?
CTCF
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what does CTCF have that allows it to bind?
Zinc finger DNA bidning protein domain
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what is CTCF?
a TF
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what does CTCF bind to?
HS5 and 3' HS1 that defines the neck of the loop in the LCR, binds to 34 bp target DNA sites with a storng consenus seq
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how many Zinc fingers has it got?
11 ZFs
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what is CTCF doing? where is it found?
found in almost every loop = its a major organising factor for 3D structure
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what does a heat map of chromatin conformation capture show?
contacts between different regions of the locus- map where so diagnoal line in middle shows blue seq always found with blue seq
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what does a darker bit on a heat map mean?
the intensity frequency of those 2 things are greater = more often seen together
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what does the heat mapping allow?
heat mapping method of chromatin conformation capture allows you to capture seqs and map all DNA in close proximity with one another = genome wide analysis of chromatin interactions
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why can you cut it in half and look at triangles?
because its a pairwise interaction it'll be the same for both
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what did they hsow with heat mapping of olfactory receptor clusters in mouse neurons?
genes within the cluster interact with one another but end of cluster interactions stop- not getting any more interaction- can cosntruct functional map of how 3D structure changes when TF absent/present or differentiated cells
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what are topologically associating domains?
higher order organisational units of chromatin function within the nucleus
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what can you work out by comparing heat maps in different cell types?
how the cell state and the TF repertoire affect higher order chromatin structure
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what do topologically associating domains do?
identify the extent of a chromatin domain containing transcriptionally co-regulated genes, TAD ends identify boundary elements that insulate genes within TADs from elements next to them
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what topologically associatd domains have you got?
TAD 1 + TAD 2
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what is CTCF doing and where is it located?
CTCF is that DNA binding protein that defines the beta globlin LCR loop in erytroblats- CTCF sites are located at the boundary between topologically associated domains
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how are topologically associating domains organised?
by CTCF binding the DNA togteher and boundary/insulator factors and cohesins making chromatin loops to ensure it regulates only its own genes
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