Gene Regulation During Development

Question 1

How do cells progressively become different from each other

Answer 1

Changes in gene expression

Question 2

Process of differentiation basic

Answer 2

Series of Hierarchical fate decisions
Before reaching a final nondifferentiatable state

Question 3

TFs to convert fibroblast to neuron

Answer 3

Ascl1
Brn2
Myt1I
Change gene expression - altering cell type

Question 4

Gene regulation - activation

Answer 4

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)

Question 5

Gene regulation - repression

Answer 5

DNA methylation
Repressive histone marks
Transcriptional repressor proteins
Transcription pausing
RNA processing
RNA destabilisation (via proteins or miRNA)
Translation (and protein modification)

Question 6

Regulation at chromatin level - histones

Answer 6

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

Question 7

Histone acetylation

Answer 7

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

Question 8

Histone deacetylase complexes

Answer 8

Histone deacetylases are part of complexes
Recruit other factors that also repress transcription
Eg NuRD complex
Sin3 complex
REST complex

Question 9

Polycomb complex

Answer 9

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

Question 10

Polycomb mutants

Answer 10

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

Question 11

NuRD component mutations

Answer 11

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

Question 12

Role of repressive complexes in development

Answer 12

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

Question 13

Epigenetic marks

Answer 13

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

Question 14

Epigenetic marks and Locking in

Answer 14

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

Question 15

Setting a threshold for lineage commitment

Answer 15

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

Question 16

Morphogen gradients and thresholds in developmental patterning

Answer 16

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)

Question 17

Regulation at transcriptional level - promoters and enhancers

Answer 17

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)

Question 18

Importance of enhancer regions

Answer 18

Important for gene expression complexity

Question 19

Regulation at transcriptional level - TF basics

Answer 19

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

Question 20

Reagulation at transcriptional level - regulatory logic for transthyretin (TTR)

Answer 20

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

Question 21

TF logic

Answer 21

Operate in combinatorial codes
Bind promoters and enhancers

Question 22

Why not just have one TF regulating each gene
Why is having multiple TFs combining to regulate one gene needed?

Answer 22

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

Question 23

TF co-opting - Hhex

Answer 23

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

Question 24

Transcriptional repressor action

Answer 24

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

Question 25

Gene regulation other than transcriptional control

Answer 25

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

Question 26

Why is regulation so complicated

Answer 26

Cells need to integrate and process info from multiple inputs

Question 27

Problem in coordinating expression of genes in different cell types

Answer 27

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

Question 28

Finding distal enhancers

Answer 28

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

Question 29

Identifying regions with particular histone marks

Answer 29

DNA-protein cross-linking An precipitation

Question 30

DNA-protein cross-linking Ab precipitation Process

Answer 30

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)

Question 31

Identifying DNA regions that contact gene of interest by looping

Answer 31

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

Question 32

Identifying DNA regions that contact gene of interest by looping if no candidate is known

Answer 32

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)

Question 33

Identifying enhancer regions - summary

Answer 33

-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

Question 34

Hex enhancer reporter assay

Answer 34

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

Question 35

Paused polymerase

Answer 35

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

Question 36

GWAS of paused polymerase in drosophila

Answer 36

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

Question 37

Genes enriched with paused polymerase in stalled Pol II fraction in drosophila PP GWAS

Answer 37

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

Question 38

How to manage the 2^20k expression states without equally as many input signals?

Answer 38

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

Question 39

“Processing power” of the TF network

Answer 39

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

Question 40

Mutual repression properties

Answer 40

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

Question 41

Mutual repression in blood differentiation

Answer 41

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

Question 42

Some combinations of TFs are more stable than others

Answer 42

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

Question 43

GRN summary

Answer 43

Properties of GRNs constrain the possible combinations of genes that can be co-expressed The logic of GRN motifs can guide cell differentiation decisions

Question 44

Enhancers and paused polymerase summary

Answer 44

Enhancers integrate info from multiple sources Paused polymerase integrates information from additional sources