Gene Regulation During Development Flashcards
How do cells progressively become different from each other
Changes in gene expression
Process of differentiation basic
Series of Hierarchical fate decisions
Before reaching a final nondifferentiatable state
TFs to convert fibroblast to neuron
Ascl1
Brn2
Myt1I
Change gene expression - altering cell type
Gene regulation - activation
DNA demethylation (permissive)
Active histone marks (permissive?)
Transcriptional activator proteins (instructive)
Transcription release
RNA processing
RNA stabilisation (via proteins or absence of miRNA)
Translation (& protein modification)
Gene regulation - repression
DNA methylation
Repressive histone marks
Transcriptional repressor proteins
Transcription pausing
RNA processing
RNA destabilisation (via proteins or miRNA)
Translation (and protein modification)
Regulation at chromatin level - histones
DNA wrapped around histone octamer of 4 core histones
H3
H4
H2A
H2B
Have n terminal tails that can be modified (acetylation, methylation)
Certain modifications correlate activation or repression
Catalysed by particular enzymes
These marks change dynamically during development
Histones keep DNA packaged
Can actively influence expression
Histone acetylation
Usually on lysine
Neutralises lysines +ve charge
Weakens histone’s interaction with -ve charged DNA backbone
Losses DNA association
Makes genes more accessible to transcription machinery
Deacetyltransferases remove the acetylation
Reinstating the interaction of lysine with DNA backbone
repressive again
Histone deacetylase complexes
Histone deacetylases are part of complexes
Recruit other factors that also repress transcription
Eg NuRD complex
Sin3 complex
REST complex
Polycomb complex
Contain enzymes that catalyse H3K27 METHYLATION
Represses transcription
By enabling recruitment of proteins that modulate chromatin and transcription
Is important for maintaining correct gene expression in differentiated cells
Polycomb mutants
Fail to establish cell identity - homely if transformations
Mine. With mutations in PRC2 component fail to gastrulate
Can’t see in embryo but mutant ES cells in dish -
ES cells are fine and can differentiate OK
But differentiated cells don’t have the correct expression profile
So mouse fails to gastrulate as differentiated cells at gastrulation have wrong gene profile so act incorrectly
NuRD component mutations
Mutant ES cells can renew but are unable to initiate differentiation unless v strong cues are given
NuRD complex important for establishing correct gene expression so cells CAN differentiate
Role of repressive complexes in development
Transcription factors initiate changes in gene expression
Then chromatin modifications stabilise these changes by enforcing on/off state
LOCKING IN
Or alternatively:
Chromatin modifiersser particular gene activation threshold
SETTING THRESHOLDS
Epigenetic marks
Epigenetic marks: accessibility of binding sites on DNA
-help regulate gene expression and can help cells remember their history
Important as allows cell to integrate information that arrived at different times
And because same signals are often Used to generate different cell types
Epigenetic marks and Locking in
2 genes In development
1st signal
Initiates gene expression change
Chromatin modification Locks In the change
2nd signal comes that can potentially activate both of these genes
But gene 2 cannot activate due to the Epigenetic marks from chromatin modification
Epigenetic marks let the same signal have a different effect in different contexts
Allows the cells to integrate into that arrived at different times because same signals often used to generate diff cell types
Setting a threshold for lineage commitment
Depending on the modification, DNA is tighter or looser
Influences access by transcription machinery
Tighter = decreased probability of transcription
So will need more transcription signal/signal to stay longer
Allows for MORPHOGEN GRADIENTS
Morphogen gradients and thresholds in developmental patterning
Different modifications in different genes
Different tight/losseness in different genes
So different reaction to morphogen signal depending on its concentration
(Only cells closest to source will have highest thresholds crossed)
Regulation at transcriptional level - promoters and enhancers
Depends on recruitment of RNA pol II
TFs bind promoter
Can help to recruit/stabilise the machinery
Can also bind long way upstream, downstream, within introns - and stil control expression (far away binding is binding at enhancers)
Importance of enhancer regions
Important for gene expression complexity
Regulation at transcriptional level - TF basics
Avtivate or repress trancription
If to specific short DNA sequences
Sequences bound can vary (unlike a restriction endonuclease)
Biding and activation/repression is cell context dependent - depends on combination of the other TFs bound there and chromatin state
Reagulation at transcriptional level - regulatory logic for transthyretin (TTR)
5 TFs control this gene (bind at promoter AND enhancer regions)
Why?
Same reason as why some bind far from promoter
Activation of TTR in liver requires all 5 Transcriptional activators
The fact that a large number of TFs are present to bind and stabilise RNA pol II (including far bound ones) allows the RNA pol II to work
TF logic
Operate in combinatorial codes
Bind promoters and enhancers
Why not just have one TF regulating each gene
Why is having multiple TFs combining to regulate one gene needed?
So that the same TF can be used for different cell fate decisions
Reason - our complexity has EVOLVED
In ancestor - one TF per fate decision
But as complexity arose TFs were co-opted for different varied functions
Eg TF1 controls fate decision between cell type A or B
But May also be needed later to choose between F or G
TF co-opting - Hhex
Early in development - controls choice of endoderm cells to become anterior endoderm
Later on controls both haematopoiesis and liver development (mesodermal)
In each of these two contexts
Hhex is working
-early on - with diff set of early present TFs and in one chromatin context
-later on with a different layer set of layer present TFs and in a different chromatin context
Transcriptional repressor action
Binds and prevents activators from binding
Binds in a separate spot to activities but neutralises it by preventing interaction with the machinery
Preventing recruitment of machinery by GTFs
Gene regulation other than transcriptional control
Post translational RNA regulation
-regulation via addition or removal of 5’ cap or 3’ polyA tail - affects transcript stability
-differential splicing - can generate multiple versions of same protein with diff domains present or absent
-microRNAs bid mRNA UTR and inhibit their translation - miRNAs are developmentally regulated: cell type specific
Why is regulation so complicated
Cells need to integrate and process info from multiple inputs
Problem in coordinating expression of genes in different cell types
Gene expression needs to be regulated in coordinated way for different developmental fates
Genes get signals from many diff sources
Problem - huge number of possible states of gene expression 2^20k - expression from 1000s genes needs to be coordinated
Cell also receives info from many outputs - needs to integrate it
Finding distal enhancers
Distal enhancers can be brought near transcription start site by bending of DNA
Can find enhancers from the TF
-look for sequence where TF binds
-look within certain range of gene (after certain range looping back is impossible) - need to look within plausible region range
-look for certain histone marks in regions
-look for regions which contact gene through looping back
Identifying regions with particular histone marks
DNA-protein cross-linking
An precipitation
DNA-protein cross-linking
Ab precipitation
Process
Stabilise interaction of DNA and proteins so they stay bound
Sonicate DNA to fragment randomly
Have Ab that recognises Hhex TF
Ab is heavy - allows precipitation of DNA attached to the Hhex protein
Sequence precipitated DNA:
Can see which bits of DNA bind the protein of interest
(TF, specific histone modifications)
Identifying DNA regions that contact gene of interest by looping
DNA FISH
When enhancer controls expression- that bit of DNA is looped around close to start site of transcription
Use fluorescent probe in. One colour to label candidate enhancer
Use diff colour to label transcription start site
Can see if these two loci overlap in nucleus with fluorescence microscopy
Limitations:
Need
To know where candidate enhancer is
Can only do for one gene at a time
Identifying DNA regions that contact gene of interest by looping if no candidate is known
Ask which bits of DNA contact gene of interest
Use 3C (chromosome confirmation capture)
-cross link to stabilise DNA-protein interactions (eg the ones keeping the enhancer looped back near the start site) to stabilise enhancer-gene proximity
-cut w restriction enzyme-obtains clean blunt ends that can be religated
-religate into small loop - creates brand new sequence at ligation site as it brings together two distant parts of genome
-ligated DNA is sonicted
-this DNA has been biotin labelled so can precipitate out all the religated fragments
-purified
-sequnce the fragments
-can map out which bits of DNA are newly interacting with each (by analysing which sequences from the genome have been fused together to form the new sequences)
Identifying enhancer regions - summary
-regions w active histone marks (ChIP)
-regions w bound TFs - ChIP and binding site prediction
-identifying regions that are in contact with gene of interest (3C, DNA FISH)
-sequence conservation - regulatory regions likely conserved between species
Above are all prone to false positives so reporter assays with candidate regulatory sequences also helpful to see if false or no
Hex enhancer reporter assay
Expressed in many parts of embryo
Where are the enhancers that drive transcription in these different regions?
-are they actually enhancers?
-if so- where do they drive expression- specific enhancers for liver, endoderm?…
Diff enhancer regions drive expression in diff tissues
Introduce reporter artificial construct into embryo (eg LacZ)
Give diff markers to diff enhancers to see which is driving expression where
Paused polymerase
Important for integration of info from diff inputs
First - having multiple enhancers helps with this
Initiation regulated independently of elongation
-a checkpoint during gene expression
-allows integration of multiple signals
-allow for rapid response- poised for activation
-allows coordinated, synchronised activation of many genes - can start up recruited machinery on many genes all at once
GWAS of paused polymerase in drosophila
High no. of gene s with polymerase right at gene start site in drisophila embryo cells compared to running along gene
Paused polymerase checkpoint
Not every gene faces this checkpoint
Genes enriched with paused polymerase in stalled Pol II fraction in drosophila PP GWAS
Genes not needed here - turned off
Housekeeping genes - being transcribed
Cells with stalled polymerase are the ones necessary in development in gastrulation
Tells us that pausing is important for development - want all the gastrulation genes to be coordinated
Other genes with paused polymerase are things like heat shock/stress proteins
Need to have rapid stress response and DNA repair too
Held in check ready to respond to signals
How to manage the 2^20k expression states without equally as many input signals?
1000 genes in human genome encode TFs
Bloated middle management in cell
Want TFs to bind regulatory regions of other TFs - regulate them
Ask what TFs does this TF regulate?- then ask what does that one regulate - often loop back round - feedback eg
Forms complicated TF network
Properties of network like computer circuit - have processing power
“Processing power” of the TF network
Focus in on small sub networks of whole TF network
Recurring motifs
Eg mutual repression
One TF repressed another
And that TF represses the first one back
Often times when this motif is seen - the TFs also upregukate their own expression
Mutual repression properties
Both TFs try to repress each other
Just need scales to tip one way - slightly higher expression of one over other
Increased expression of green:
-cause increased repression of red
-autoamlification of green is increased
-autoamolficatuon if red also decreased due to increased red repression by green
-red goes down
-so less repression on green
-green goes up
-more repression of red, more autoamllification of green, less of red
Feedback Leads to cell collapsing into state of just having green
Can control differentiation - where green controls downstream all the genes for one cell type, and red another
Both of these TFs can’t be stably expressed at same time
Eg in blood
Mutual repression in blood differentiation
Gata1-Pu.1 cross antagonism
In blood differentiation in bone marrow - it would be difficult to control differentiation with just spatial cues
So mutual repression mechanism is also part of differentiation control
Some combinations of TFs are more stable than others
Eg
2 TFs
Both on is more stable than only one or the other on
So out of the 2^20k states - only much fewer if then are actually stable for the cell to be in
So when generating a cell type in the body - means that you don’t need to regulate all of the individual genes
-just need to push a few TFs in the right direction
-and the properties of the state will get it into this cell type
GRN - gene regulatory network
GRN summary
Properties of GRNs constrain the possible combinations of genes that can be co-expressed
The logic of GRN motifs can guide cell differentiation decisions
Enhancers and paused polymerase summary
Enhancers integrate info from multiple sources
Paused polymerase integrates information from additional sources
