V) Memory Engram At Epigenetic Level

Question 1

How does an engram forms ?

Answer 1

Groups of interconnected neurons are activated together during learning and their récurent and stimulateous activation during memory consolidation produces a strengthening of the synapses between them, which determine THUIR réactivation during memory recall.

Question 2

What is the results of cellular assembly during learning ?

Answer 2

Synaptic plasticity and reshaping of neural circuit

Question 3

Cites the molecular and cellular changes in engram cells

Answer 3

Stimulation
Cascade of events
Neurotransmitter release & binding to membrane receptors
Rise of Post synoptic ca2+
Activation of ca+ dependent pathway and TRANSCRIPTIONAL EPIGENETIC CHANGES which contribute to synaptic plasticity including LTP

Question 4

Which type of responsive genes are inducted for plasticity

Answer 4

Immediate early genes (IEg) - transcription factors (e.g fos)
Late response genes (LRg) effector of plasticity

Question 5

Site différents transcriptional and epigenetic changes in engram cells

Answer 5

Induction of plasticity related genes
Chromatin remodelin

Question 6

4 types of chromatin remodeling

Answer 6

Histone poste translational changes
DNA methylation changes
3D chromatin architecture remodeling
Enhancer transcription (eRNaS) activation

Question 7

Cite recents development of advanced functional genomics

Answer 7

Fos Trap system
Cell type specific omics analyses including single cell transcriptomics

Question 8

Who are the actors of the histone code ?

Answer 8

Writers: to frite the code (acetylation, methylation)
Readers: to interpret the code and convey the information in functional outcome
Erasers to dynamically regulate the code and adjust to cellular state/adapt to cellular environment or stimuli

Question 9

Are there specific histone code ?

Answer 9

Brain specific modifications: histone serotonylation, dopaminylation
Role and mechanism remain elusive

Question 10

Sites active histone marks

Answer 10

H3k4me1/3
H3k9ac
H3k27ac

Question 11

Répressive histone marks

Answer 11

H3K9me3
H3k2me3

Question 12

How is the 3D chromatine architecture

Answer 12

Different levels and scales of organization
Defined organization
Dynamic regulation

Question 13

What is the advantage of chromatin loops ?

Answer 13

They allow the physical interaction between promoter and enhancer (regulate transcription)

Question 14

What are TaDs

Answer 14

Topologically associating domains (large self interacting chromatin region)

Question 15

How enhancer RNAs are synthesized at active enhancers and positively regulate their target gene ?

Answer 15

Stabilisation of enhancer/promoter chromatin loop
Stimulation of histone acetylation
Stimulation of transcription elongation

Question 16

Rôle : RNA-seq

Answer 16

Transcription profiling

Question 17

Rôle : ATAC-seq

Answer 17

Chromatin accessibility

Question 18

Rôle of ChIP-seq)

Answer 18

(histone modifications, transcription factor binding profiling

Question 19

DNA seq role ?

Answer 19

DNA methylation

Question 20

Hi-C

Answer 20

3D chromatin architecture

Question 21

RNA seq steps

Answer 21

RNA extraction
Library preparator
Sequencing
Data analysis (differential or functional)

Question 22

ATAC seq steps

Answer 22

Atac
Library preparation
Sequencing
Data analysis

Question 23

Chip seq steps

Answer 23

Chromatin cross linking
Chromatin sonication and immuno precipitation
Immunocomplex purification
DNA elution and purification
Sequencing

Question 24

Chromosome conformation capture ?

Answer 24

Capture interacting regions through cross linking, digestion, ligation and PCR/sequencing. The anchor and potential interacting regions are known.
(Fixation, digestion and ligation)

Question 25

Cites cell type specific NGS technologies

Answer 25

Cell RNAseq
Nuclear RNAseq

Question 26

Which techniques for single cell analysis of the epigenome

Answer 26

ATACseq at single cell level (snATACseq)
DNAseq -> snRRBS
HiC at single cell level : snHi-C
CUT&Tag (similar to ChIpseq) at single cell level : snCUT&Tag

Question 27

Limits of single cell analyse ?

Answer 27

Extensive costs
Data analysis challenge
optimization required, remains a challenge

Question 28

By what are triggered transcriptional and epigenetic changes associated with neuronal activation in neuronal culture ?

Answer 28

They are induced by neuronal activation

Question 29

How has been shown regulation at IEgs ?

Answer 29

Collection 1h to 6h after KCL stimulation and control (w/out KCL)

RNA seq shows activation of Fos gene 1h after kcl

Fos induction is accompagniez by activation of distal regulatory elements (enhancers) - shown with chIp ses

Question 30

Explain regulation of LRGs

Answer 30

ChIp seq for many ieg (among them: fos) binding. Increased CBP and increased H3K27ac at target promoter and/or enhancer.

Question 31

What are shRNA

Answer 31

Short hairpin RNA to reduce RNA expression c:

Question 32

What effect when shRNA fos on transcription of fos target ?

Answer 32

Reduced activity of regulation of fos targets.

Question 33

What is the composition of engrams ?

Answer 33

First experience cells
Second experience cells
Reactivated cells

Question 34

How to use NGS to tag cell in vivo?

Answer 34

Fos Trap / arc Trap (targeted recombination active population) : Long lasting labeling of cells activated during learning to Id long term memory engram cells: td tomato expressed when activity and tamoxifen .

Active cells that express CreEr undergo recombination when tamoxifen is present. In active cells do not.

Question 35

Site the epigenetic event related to memory encoding

Answer 35

Increased accessibility of subsets of enhancers without transcriptional changes

Question 36

Site the epigenetic event related to memory consolidation

Answer 36

Spatial chromatin reorganization and new promoter-enhancer interactions leading to transcriptional activation of LRG promoting synaptic plasticity

Question 37

Site the epigenetic event related to memory recall

Answer 37

Spatial architecture of the chromatin is firther reorganized, additional promoter enhancers interactions and activation of genes notably implicated in protein translation, thereby contributing to synapse re organization.