Epigenetic regulation of brain development and function

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  • Created by: Sarah
  • Created on: 24-05-19 12:31
what does the brain allow us to do?
make sense of and interact with the world around us, receives processes+integrates info from environmental sources and body, transmits info to diff body parts+ regs behaviourhal interactions with environment, adapts its functions
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why does the brain improve physiological performance and optimise behaviour?
it adapts its own functions and enables other parts of the body to adapt in the light of information
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when are brain circuits esatblished and activated?
during embryonic development
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how are brain circuits elaborated and modified?
by experience-sensitive molecular mechanisms
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changes in synaptic activity can induce what?
changes in synaptic strength, the voltage change elicited in post synaptic neurons
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why is the voltage change/synaptic strength altered because of?
altered expression or funciton of NT receptors (glutamate AMPA receptors)
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what are the changes in synaptic strength called?
long term potentiation and long term depression
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how is LTP maintained?
by altered transcription and epigenetic modification of genes involved in synapse function and memory formation and persistence
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Describe the role of LTP in long term memory formation?
glutamate bind to post-synpatic rec -> ca influx -> CamKII activation -> CREB TF phosph -> CRE-containing promoter activation -> altered synapse gene expression -> altered synapses
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what are CRE-containing promoter genes?
genes involved in memory formation
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what causes a calcium influx into the post-synaptic neurons?
synaptic activity
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what does synaptic activity-induced ca influx cause?
phsophorylation of CREB which activates it, is a TF that moves into nucleus leads to neuron-specific, synaptic activity-dependent gene transcription
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how did they show this?
stimulates the neurons with Kcl and saw more Phosphorylated CREB proteins with fluroscent antibodies and more Npas4 00000protein
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what is Npas4?
one of the synaptic activity-dependent genes, a downstream target of CREB TF
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when we're thinking about embryonic development- processes that establish brain circuit are flexible hiw?
epogenetics- circuits can be structurally elaborated or reduced in scale and functionally altered as a result of molecular mechanisms that can be encountered in utero, after birth and throughout life
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what is LTP?
the phenomenon where neuronal activity itself elicits changes in synaptic strength that can be increased due to changes in num of Nt receptors and NT rec function in the synpase
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what's it called when synaptic efficacy increases (increase in synaptic strength)?
LTP
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what's it called when synaptic efficacy decreases?
LTD
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Describe the overall change in LTP
small num of glutamate recs on the postsynaptic neuron -> after neurotransmission glutamates been released and postsynaptic neuron changes the properties of the synapse so increased level glutamate recs activity more readily trigger when next NT rele
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what is LTP maintained by?
altered transcription and epigenetic modifications of genes involved in synapse function and memory formation/persistence
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what is already synthesised and stores of in the neuron so they're ready to respond and be recruited and inserted into the synaptic membrane when an excitatory neurotransmission occurs?
stores of glutamate receptors already synthesized
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what is there also de novo synthesis of?
gene products as a result induced gene transcription in the nucleus that the presynaptic neuron engages with (in the postsynaptic neuron nucleus)
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what is a critical TF that responds to neural activity
CREB
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what does CREB stand for?
cyclic AMP response element
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what does CREB respond to? where?
in the postsynaptic neuron CREB responds to calcium influx triggered by glutamate binding, ca influx -> calmodulin dependent kinase 2 (CamKII) phos CREB so it moves into nucleus as TF drives transcription of genes
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what happens to mouse cultured quiscent neurons when you add Kcl?
they will spontaneously depolarise
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what will happen if you spontaneously depolarise mouse neurons with Kcl?
phosphorylated CREB is very rapid consequence of this stimulus as see an increased expression of a number of CREB target genes
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what CREB target genes do you see an increase in?
Npas4- a basic-helix-loop-helix TF
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what experiment did they do to show CREB response genes were expressed after Kcl stimulation?
did a WB- Npas4 came on rapidly within 2 hours of exposure to Kcl
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what is another gene that is rapidly transcribed in the WB after Kcl depolarisation?
cFos
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what else is on the WB more of?
phosphorylated CREB itself
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what else will turn on genes like cFos and Npas4?
BDNF, Neurotrophin 3 and 4- are induced by a variety of stimuli, neurotrophic IC signalling proteins
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what is induced in response to neuronal depolarisation by Kcl?
CREB is phosphorylated and Npas4 and cFos are induced
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what is pentylenetetrazole?
a convulsant agent
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what does the pentylenetetrazole convuslant agent do?
is a synaptic activity-inducing compound
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what happens when you expose ZF larvae to a synaptic activity-inducing compound like pentylenetetrazole?
induced transcription of synaptic activity-related gene expression
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what synaptic activity-rregulated genes are turned on if you induce synaptic activity?
cFos and Npas4 in a few minutes expression is increased then later BDNF is induced
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what is BDNF induced by?
neural activity- is a neural activity-induced signalling protein
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what is BDNF required for?
activity-induced enhancement of neuronal survival, axon outgrowth and arborization
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in response to neural activity there is very poweful activity dependent transcriptional change in the brain what is the function of this response
in part to remodel neuronal and axonal morphogenesis
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if you take neruons in culture and treat them in culutre what happens?
they depolarise and produce dendrites and interact more with one another
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what increases with Kcl stimulation of neurons?
increased extensive network of interacting neurons
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what happens to the outgrowth of dendrites and interations if you use an antibody against BDNF which is activity-induced?
axonal arborisations and synaptic connections fail to develop in these cultures- the role of synaptic activity-dependent gene treanscription is at least in part o facilitate remodelling of neuronal circuitry improves arborisation and synaptogenesis
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what part of the brain is largely important in learning activities and memory processes?
hippocampus
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what is the contextual fear conditioning paradigm?
animal taken from home cage put in a new cage with a stimulus like a shock, new cage has context like striped floor, associate stripe floor with shock, return to homecage, put in context so stripe floor and will respond with shock response (freezing)
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step 1 of contextual fear conditioning?
mouse + training cage + foot shock -> mouse freezes and associates cage with shock = memory acquisiton
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what happens in step 2- memory consolidation of contextual fear conditioning?
mouse returned to homecage (reflects on experience)
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what happens in memory retrieval, step 3, of contextual fear conditioning?
mouse placed in context cage but without shock- memory retrieval, will remember the context having the shock and will freeze even though no shock
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what is measured?
freezing duration elicited by context without the shock
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what are you pairing in the contextual fear conditioning?
the context (stripey floor) + the shock- do this several times
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what happens when you shock a mouse?
it will freeze- physically immotile then recovers
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what is the freezing behaviour a physical manifestation of when presented with just the context alone?
the creation of the memory of the context and shock
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what is the period of memory acquisition?
the process of repeated pairing of context + shock
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whats the period of time when they reflect on their experience?
memory consolidation
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how did they measure the memory existence?
presented only the context and measured the behavioural response- amount of freezing thats inhibited in retrieval period
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what did DNA methylartion in the rat hippocampus do to memory cosnolidation?
enhanced memory cosnolidation
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what showed an increase level of expression after animals were subjected to this process and presented to the coneext alone?
increased expression of de novo DNA methyltransferases DNMT3a
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what did they find if they treated the animals with a DNA methyrransferase inhibitor?
they found the ability of the animals to remember the shock in the context alone was reduced
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what did this imply?
that the DNA methyltransferases were required to recall the memory of the shock
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what genes did they find that might be mediating these effects?
Protein phosphatase 1 (PP1)
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when was PP1 expression increased?
when they gave the DNA methytransferase inhibitor- PP1 expression was increased (so supresses memory)
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what kind of gene is PP1?
a tumour supressor gene- is increased when you can't recall the memory
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what DNA methyransferase inhibitor did they use?
5-azacytidine
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what happened in the animals treated with 5-azacytidine?
increased expression of PP1 and can't recall shock when presentedf with context
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what does this imply?
that DNA methytransferases are involved in memory acquisition by inhibiting genes that supress memory (like PP1)
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what is calcineurin involved in?
regulating the level of phosphorylation of CREB in response to neuronal depolarisation and ca influx
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what does memory consolidation cause an increase and decrease in?
memory consolidation caused increased promoter DNA methylation and decreased transxription of the LTP learning and memory regulator caclineurin
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where is calcinuerin transcription reduced?
in the rodent prefrontal cortex
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what increases over time as the context and shock get co-administered?
methylation of the caclinuerin promtoer
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what happens to calcinuerin level of transcirption in emmory consolidation and remembering the shock with the context?
DNA methylation of calcinuerin is increased, level of transcription is decreased
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what is calcinuerin normally limiting?
recall of that memory (the shock and context)
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when they used 5-azacytidine (DNA methyltransferase inhibitor) and other inhibitors were all doing what
reducing the level of freezing when the context was presented alone
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what does that suggest DNA methylation is doing?
need the methylation without it they don't remember so needed for memory acquisition
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how will memory supressors be affected with the DNA methytransferase inhibitors?
DNA methylation inhibitor -> memory supressors won't be methylated -> memory supressors supress memory -> don't freeze in context
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what happens to caclineruin in memory consolidation?
calcineurin promoter becomes methylated, less transcription so memory supressor genes are inhibited
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what do TET enzymes do?
are demethylases- remove methylation marks
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how can DNA methylation patterns be ACTIVELY and specifically removed from the genome?
by TET enzymes- multistep methyl-group removal and repair process
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how do TET demethylases work?
cause hydroxyl methylation of methyl-cytosine leading to the removal of the base, base excision repair producing unmethylated cytosine
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is the TET enzymes removal of methyl marks an active process?
yes an active process
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what happens in TET1 KO brains?
increased promoter methylation is accompanied by reduced synaptic activity-regulated gene transcription
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what is TET1 required to promote?
the activity of synaptic activity-regulated genes
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when do methylation levels of synaptic activity-regulated genes become very high?
in the TET1 KO
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what gene can this be seen with?
increased methylation across Npas4 (immediate early activity-induced TF) - whole locus is hypermethylated
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where is this methylaiton increased in the TET1 KO?
in the cortex and in the hippocampus
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what is the WT methylation of Npas4?
no methylation across the gene
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is this hypermethylation in TET1 KO true for other synaptic activity-regulated genes?
yes true for a number of genes
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what do you see along side the methylation?
a reduction in transcription of the synaptic activity-regulated genes
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What is increased and decreased with the loss of TET 1 demehtylase activtiy?
So gene transcription is reduced, methylation is increased as a result of the loss of this active demethylase activity
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what happens to TET1 KO in the contextual fear paradigm?
ability to form memories not impaired freeze in response to context without shock but if you put them in their homecage or another cage then WT animals dissociate link over time memory is extinguished. Exctition is prevented can't forget in TET1 Ko
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what does the phase of extinction result in?
the level of freezing when the animals are presented with the context alone decline
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what do TET enzymes doing here?
promote demthylation (memory supressors expressed) and facilitate fear extinction memories
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what happens in a WT mouse with fear extinction?
over time freezing diminishes in response to the context
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what do the TET1 KO have persitently high levels of?
recall- they're unable to extinguish the memory
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what does this say about the methylation patterns laid down in memory formation?
as the memory is lost the methylation is lost too
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what did the transcriptome analysis of the cortex + hippocampus show when they compared the TET1 KO and WT animals?
many of the neural activity-regulated genes are downregulated in the TET1 KO
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why are these genes likely to be downregulated in the TET1 KO?
in the TET1 KO methyl marks not removed so there's excess methylation like with Npas4 so downregulated
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what are TET enzymes required for in memory?
demethylation of genes so that you can have memory extinction (so you can forget)
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what is there a good association between?
failure to extinguish the memory and failure to erase the DNA methylation patterns from activity regulated genes
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what do you probably need for memory extinction?
DNA demethylation process- need TET enzymes (think of it like demethylate express memory supressors so you can't think of the memory anymore)
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all these activity regulated genes do what in the TET1 KO?
level of methylation is increased and their expression is decreased
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what has been focused on for the role of histone modifcations in memory?
HDAC2 - histone deacteylase 2
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what does histone deacteylase 2 do?
removes acetyl marks from chromatin
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what happens if histone deacteylase 2 is overexpressed?
level of freezing in fear contextual paradigm is reduced
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what does histone deacteylase 2 do to memory?
it supressed the memory consolidation and the neuronal dendritic spine formation in the mouse brain
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what happens if you overexpress histone deacteylase 1?
nothing- so this is specific to HDAC2
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What happens if you KO HDAC2?
power of recall is enhanced- stronger memories of the shock and context
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what does this suggest histone deacteylase 2 is doing normally?
limiting memory formation (as when you remove it then recalls increased)
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what happens when they looked at the individual dendritic spines forming on neurons within the brain?
KO HDAC2- increase in amount of dendritic spine formation, overexpress HDAC2- wipe out dendritic spines
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how did they see the outline of individual neurons to look at dendritic spines?
golgi stain
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what does this suggest there's a direct link between?
neuronal structure and power of memory recall
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what did chromatin immunoprecipitation of brain tissue with anti-HDAC1/2 antibodies show?
HDAC2 specifically binds to the promoters of neuronal genes implicated in the regulation of synapse function and memory, many synaptic activity-regulated genes associated with HDAC2 protein
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what are the different sets of genes?
neuronal activity-regulated genes involved in synapse formation (proteins to make synapses) or genes involved in synaptic plasticity LTP and LTD
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What do all these genes bind on the pulldown?
HDAC2 is actively on these locci and antagonising acetylation of synaptic activity-regulated genes
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what do you see happen to acetylation marks in the HDAC2 KO?
look using antibodies that recognise acetylated H4 and acetylated H3 when deacetylase KO there's an increase in acetyl marks on a subset of those activity-regulated genes
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what HDAC inhibitor was used?
SAHA
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What does the HDAC inhibitor SAHA do?
enhances memory consolidation (of the fear), rescues memory and dendritic spine formation deficits caused by HDAC2 overexpression
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what happens if you take animals that have been through the contextual fear conditioning paradigm and assay how much they freeze with SAHA?
with SAHA that increases freezing- increases memory to recall the memory
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what does overexpressing HDAC2 do to memory?
reduces memory recall
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what happens if you overexpress HDAC and then give SAHA
increases memory
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what does SAHA do to HDAC2 Ko?
nothing because it can't inhibit HDAC if its KO
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what happens to dendritic formation in animals that received SAHA either WT or HDAC2 overexpression?
SAHA- restores dendrites or increase number s of dendrites in HDAC2 over expressing or WT
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what is increased acetylation of neuronal synaptic genes doing to dendritic spine formation and memory formation?
acetylation -> impairs dendritic spine formation and memory formation
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what is the key acetyltransferase in this process?
CBP- crucial role of neural activity- dependent gene regulation
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what does CBP acetyltransferase bind to
CREB and potentiates activity by locally acetylating target genes
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Heterozygosity for a mutation in the human CBP gene, or the close relative P300 causes what?
severe disorder = Rubuinstein-Tabyi Syndrome (RTS)
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Humans that are heterozygous for LOF mutation in CREB binding protein (CBP- that acetyltransferase) have what?
a severe developmental abnromality called Rubuinstein-Tabyi syndrome where thee's multiple NS abnromalities
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what part of memory is CBP required for?
memory consolidation
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what does CBP to give memory formation?
promotes H2B acetylation and facilitates transcription of immedaite- early activity- regulated genes like cFos
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what does CBP do to CREB activity
potentiates its activity by binding to it
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what do CBP mutants show?
significant reduction in histoen 2B acetylation because these histones are dependent on CBP function
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what do CBP mutants have a reduced capacity to do?
retain memories in the contextual fear conditioning paradigm
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how do they know this?
when they looked at the freezing response when presented with context alone after they've been conditioned they will respond with a substanital amount of freezing but if hetero for CBP mutation then no memory recall
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why aren't they able to remember in CBP mutants?
lost their ability to acetyklate and activity some of those synaptic activity dependent target genes
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what was found with chromatin immunoprecipitation analysis of the cFos gene in a ***** of brain stained with an antibody against cFos?
cFos is expressed in the WT but gone in the mutant heterozygote CBP
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what did they find when they look at the cFos locus using chromatin immunoprecipitation to look at the distribution of marks and CBP?
taken neurons from animals put them in culture + stimulate with Kcl- stimulate synaptic activity- see cFos 2 enhancer elements become hyperacetylated so gene becomes transcribed
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what do we see at the same positions of cFos being transcribed?
the accumulation of Histone H3 lysine 4 methylation marks created by trithorax proteins
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is the H3K4 methylation marks activity induced?
no but its in the vincity only one of the acetylation peaks are activity induced
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what else is localised to these enhancer elements?
CBP and DNAase I hypersensitivity sites and H3K4 methylation marks- all collaborating to turn on this gene
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what do cFos and other neuroanl activity-regulated TFs bind to?
enhancers of neuronal hgenes whose activity-induced transcription requires cFos function
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all of the activity regulated genes not just cFos, like Npas4 respond in the same way to Kcl depolarisation in culutre?
by induction of synaptic activity hyperacetylation of regulatory elements which involves recruiting CBP (acetyltransferase)
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neuronal specific genes whose activity depends upon CBP function- enhancers are hyperacetylated and associated with the binding of activity regulated TFs their transcription depends on what?
the function of one or more of these activity-dependent regulated TFs
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what requires cFos function?
cFos protein and other neuronal activity-regulated TFs bind to enhancers of neuronal genes whose activity-induced transcription requires cFos function
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what happened when they added Cfos shRNA?
activation and transcription of genes is severely compromised- so the transcribability of these genes depends upon the recruitment and function of the activity regulated TFs whose trans depend upon CBP histone acetylation
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what do memory formation and extinction involve?
regulated changes to DNA methylation patterns across the genome
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what do DNA methylation by DNA methytransferases do?
promote memory formation
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what do TET demethylases do?
TET1 demethylase promotes memory extinction
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what is histone acetylation by CBP required for?
to promote memory formation- increases activity of CREB
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what does histone deacetylation by HDAC2 do?
inhibits memory acquisition
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what does administration of HDAC inhibitors do?
overcome HDAC2 activity and strengthen memory
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