Lecture Exam 1.3-1.4

experimentation has showed us that unneeded cells are

NOT lost as differentiation occurs, they simple are not used

Altough diff types of cells are needed to express unique sets of genes,

all cells came from the same fertilized egg, and so largely have the same genome

What is the basis for cell differentiation/specialization

differential gene expression

gene expression at the level of _____ is vital for early developmental changes

transcription

What are some physical ways differential gene expression occurs

• histone modifications

• DNA methylation

• Chromatin 3D architecture

• all changes chromatin to make certain areas more/less available to be transcribed

diff cells express diff sets of ______

TFs, which in turn bind to different enhancers/silencers in other genes to turn certain genes on or off

histones

proteins that help organize chromatin

• nucleosomes contain 2 of each of the 4 types of histone proteins, to form a 8 protein total complex/octamer

histone modifications

• if histone tails are methylated and deacetylated, it promotes condensed chromatin

• when the histone tails are mostly unmethylated and acetylated, it promotes opened up and decondensed chromatin

histone tails get methylated on histones ____

3 and 4

• promotes condensation

HATS

histone acetyl transferases

• adds acetyl groups onto histone 2,3,4

• promotes chromatin decondensation

histone demethylases

remove methyl groups from histones, promoting decondensation of chromatin

HDACs

histone deacetylases

• remove acetyls from histone, especially at histone tails 2,3,4

• promotes chromatin condensation

HMATs

histone methyltransferases

• adds methyl groups onto H3, 4

• promotes chromatin condensation

What promotes chromatin decondensation?

• adding acetyls (HATS)

• removing methyls (histone demethylases)

What promotes chromatin condensation?

• removing acetyls (HDACs)

• adding methyls (HMATS)

Regarding histone methylation/acetylation, acetyl groups (neg) get added to the ____

amino group of certain lysines (which are pos) in the histones

• this forms a neutral complex

which histones get methylated/demethylated

3,4

which histones get acetyled/deacetylated?

2,3,4

DNA methylation of regulatory regions in genes typically…

blocks transcription

• in vertebrates, many cytosine bases in CG pairs of the DNA sequence are methylated, forming 5-methylcytosine

• when DNA replication occurs, only the parent strand remains methylated, so it is deemed hemi-methylated

• can then be methylated again at the C of a CG, can block transcription of certain genes

How does DNA methylation actually block transcription?

• methylation of enhancers or promoters can block TFs from binding to the sequence

• methylated DNA can recruit histone modifying enzymes, which promote chromatin condensation and inhibit transcription

What happens when enhancers or promoters get methylated

• can block TFs from binding to the sequence

• ex: in globin gene switching, so that fetus has diff globins

• unmethylated regions get transcribed as usual

What happens when methylated DNA recruit histone modifying enzymes

• histone deacetylase or HMAT could be recruited

• promotes chromatin condensation and inhibit transcription

coding region of a gene

contains the info to actually make a protein (the exons) as well as introns, which are non-coding regions

what are some regulatory elements in gene expression

• promoter

• promoter proximal elements

• enhancers

• silencers

which two regulatory elements on genes can be found anywhere?

• enhancers and silencers

RNA polymerase assembles where?

at the promoters

• and the promoter proximal element goes right in front of it (goes just upstream of the promoter)

promoters

conserved sequences near the transcription start site, where RNA poly assembles to help initiate transcription

• ex of a promoter: the TATA box

• promoters are position dependent

if an enhancer is deleted,

then the gene will could not be transcribed

enhancers bind to

TFs that promote transcription

silencers bind to

TFs that inhibit transcription

position-independent regulatory dequences on genes

• enhancers and silencers

TFs

proteins that bind to regulatory regions of DNA and influence RNA Poly’s ability to initiate transcription

basal/general TFs

bind to the promoter itself

• so are similar in diff cell types

• ex: TBP (TATA-binding protein), TFIID

regulatory TFs

bind to promoter-proximal elements, enhancers, or silencers

• so vary for each cell type

• expressed depending on what genes a cell needs at a given pt in time

a single type of regulatory TFs could regulate…

multiple genes

• as long as the genes have the correct type of regulatory elements for the TF to bind to)

related genes can be turned on as a set because

a single type of TF can regulate multiple diff genes

How do activating regulatory TFs work

• promote transcription by recruiting histone-modifying enzymes that relax chromatin

  • so would recruit histone acetylase (HAT) and histone demethylase

• OR could recruit chromatin-remodeling complexes, which displace histones/reposition DNA in nucleosomes to make certain sequences more accessible

• OR could promote assembly of RNA Poly onto the promoter

chromatin remodeling complexes

reposition nucleosomes relative to DNA to make certain parts of the genome more accessible

Activating regulatory TFs promoting assembly of RNA Poly onto the promoter

uses a mediator complex of ~ 12 proteins to get RNA Poly onto the promoter to promote transcription of a certain part of the genome

mediator

a complex of ~ 12 proteins

• regulatory TFs bound to enhancers bind to mediator (a complex) to help position RNA Poly onto the promoter

diff combos of regulatory TFs can bind to diff

enhancers or silencers, allowing cells to express diff sets of genes

if the same gene is needed at multiple diff times in development,

• so diff parts of the gene must be regulated independently for formation of each separate tissue as the embryo develops

• ex: using diff enhancers to promote brain vs limb development

• this whole idea can also be used in gain of function Exps

3D organization of chromatin

helps stabilize patterns of gene expression once they are established

• ex: TADs (topologically associating domains) , which change topology of the nucleus

TADs

topologically associating domains

• proteins that help change the topology of DNA so that certain regions are more or less accessible

• allows for multiple loops of DNA to be regulated simultaneously, so genes pulled into a common TAD are regulated together (usually)

Types of TADs

B-type

• TADs that are in a region that is being repressed (inhibited from transcription)

• if a B-type TAD is stuck on a nuclear lamina, it is called a LAD (lamina associated domain)

A-type

• TADs that are in a region that is actively being transcribed, so the chromatin is less condensed

CTCF

protein that binds to sites at the boundaries of a TAD’s area

• binds to the DNA itself and stops cohesin from sliding, therefore determining the endpt of a TAD’s area

cohesin

loops the DNA between 2 TAD regions, forming chromatin loops (topologically pulls out the chromatin)

if CTCF sites between an active and inactive TAD site are deleted, then

the active TAD site expands as the CTCF site is no longer restricting it

if a CTCF site is added to an active TAD region then

that TAD region will get shortened

differential gene expression

a consequence of different cells, or the same cell at diff pts, having :

• unique patterns of chromatin structure,(diff TAD structure)

• epigenetic mods

• and expressing unique sets of regulatory TFs

differentiation is a ____ process

slow

What is the first step in studying differentiation

fate mapping

• you stop at diff pts in development and take image so see where things traveled to, and after what amount of time

• allows you to form a slideshow of what gives rise to what

_________ is what showed us that at progressively later stages in development, differentiation is a gradual, and almost always ONE-WAY process

fate mapping

attributes of fate mapping

• highly reproducible

• is very similar across diff species

differentiation is almost always unidirectional, except..

• amphibians/reptiles regrowing tails, limbs for wound healing

totipotent

cells that could become anything

• the fertilized egg

• some of the earliest blastomeres

pluripotent

cells that can become almost anything

• so can give rise to any cell an organism needs, just cannot make a whole new organism

multipotent

cells that can become a few other types of cells

• includes many of adult subsets of cells, like adult stem cells

unipotent

cells tah can only become 1 type of cell

reprogramming

the name for when you study differentiation reversal

2 phases to the differentiation process

• commitment

• cell memory

committment

when cells acquire the pattern of gene expression necessary for a specific differentiated state

• occurs during embryogenesis when a new, unique pattern of gene expression has been established

cell memory

when a cell maintains the pattern if gene expression necessary for that differentiated state

• via:

  • pos feedback loops

  • DNA methylation

  • changes in chromatin state

commitment occurs before

cell memory

RNA sequencing to study the commitment process

• take embryos at a certain stage

• break their tissues down into individual cells

• inject barcodes reagents into the cells

• the cells then release RNA which you isolate using reverse transcriptase

• repeat for thousands of cells until you have a bunch of data

• then you can see what gene gets expressed when you inject diff barcodes

• so you can see how similar gene expression is in diff cells and what comes from what gene

how could you study changes during commitment at the population level?

• by doing RNA sequencing for a few individual cells

• see how cell-level gene expression changes over time during commitment

• shows you for a given endpt, what patterns of expression occurred before it

• could do LOF Exp to see what the genes then actually control

commitment heavily depends on

the proper extracellular signals

2 experimentally defined benchmarks during commitment

• specification

• determination

specification

the pt at which a cell is far enough along a differentiation pathway that is no longer dependent on outside signals telling it to continue on that path

determination

the pt at which a cell is far enough along a differntiation pathway that it will ignore any signals telling it to choose a diff path

determination occurs ____ specification

after

How to test for specification

• culture a cell in non-biased solution to see what happens to the cell without any outside input

• if the cell type continues as before, it was already specified

How to analyze timing of commitment

• use specification map to see that state of differentiation at a given pt in time

• then compare to fate map to see where certain things specified into a certain cell type

• by comparing the 2, you can see what came when, and from where

• so shows us the timing of commitment

How to study determination

• use heterotopic (diff-place) transplantation or co-cultures

• heterotopic transplantation is when you take tissue out of one part of an embryo and graft it into another part of the embryo

  • if the cells begin to match the cell type of its new location, it was not determined

  • if the cells grow as they were before and do not change, then they were already determined

fate vs specification vs determination image

2 modes of specification in early development

• autonomous specification

• conditional specification

autonomous specification

• uncommon in vertebrates, common in invertebrates

• invertebrates usually produce many more embryos that have shorter lifespans, and autonomous specification is much more rapid

• when specific mRNAs needed for diff cell fates localize to a certain area on a cell

• so when that cell divides during mitosis, its two daughter cells are non-identical, leading to differentiation

conditional specification

• common in vertebrates

• takes longer, as does vertebrate development and lifespans

• makes vertebrate development more robust

• in conditional specification, differentiation is caused by differential exposure to key signaling molecules

• this is why wounded vertebrates can repair themselves with cells that adopt to the same fate they would have in the undamaged tissue

conditional specification allows for

compensation/ regulative development

• since cells can grow back and specialize into the cell type of the previous tissue (before the wound)

induction

• causes conditional specification

• when cells causes other nearby cells to undergo a certain developmental pathway

Induction of the lens by the optic vesicle

• example of induction

• the optic vesicle induces the surface ectoderm to thicken and form the lens placode during vertebrate eye development

• the cells must differentiate in order to form the lens

how could you study GOF or LOF with vertebrate lens formation?

LOF: ablate one of the organisms two optic vesicles and leave the other to analyze the difference

GOF: graft an optic vesicle into a diff embryo to see if it develops in a normal manner

competency

the idea that only certain tissues possess the ability to respond to a particular inducing signal (ie are competent)

• because not all tissues express the right receptor to see every signal that exists

competency in optic vesicle formation of vertebrates

• see how in GOF study, when optic vesicle is implanted into cervical (neck region), since the cells implanted lack the competency to specialize, no induction occurs, and a lens does not form

• but if you implant into the head region, the implanted vesicle would possess competency and a lens would form per usual (diff tissues respond to diff signals is what this shows)

• so only ectoderm in the head region can respond competently to the lens inducing signal from the lens vesicle, but areas outside cannot

• the unimpaired vesicle forms per usual as it possess competency

BMP4

• a lens inducing signal

• we know bc BMP4 knockout mice has optic vesicles but no lens (LOF study)

  • if you collect optic vesicles from BMP knockout mice and graft in beads soaked in BMP4 protein, then the mice would product ens per usual

• we see BMP4 is expressed in one of the arms of the optic vesicle

• if you grafted bmp4 positive vesicle into the cervical region of a mouse, no lens would form because those tissues lack BMP4 receptors

What if you grafted bmp4 positive vesicle into the cervical region of a mouse

no lens would form because those tissues lack BMP4 receptors

• bc head region expresses the Pax-6 protein, which is a TF that regulates many genes, including ones that encode receptors for the BMP family

• so a mouse embryo would need

Pax-6 expression determines

the competence of head ectoderm to respond to lens-inducing signals (like BMP4)

• we learned through combining knockout and wildtype mice’s vesicles and ectoderm that you need a wildtype ectoderm for lens induction to occur, regardless of Pax-6 knockout of wildtype vesicle being implanted, because it is the ectoderm that expresses the Pax-6

in regards to pax-6 expression in mice lens formation, if you implant wildtype optic vesicles into the surface ectoderm of a wildtype mouse, thenq

lens induction would occur per ususal

in regards to pax-6 expression in mice lens formation, if you implant pax-6 negative optic vesicles into wildtype mice surface ectoderm, then

a lens would grow, but would it would look odd

• shows us that Pax-6 is not needed from the optic vesicle itself, but from the surrounding ectoderm

in regards to pax-6 expression in mice lens formation, if you implant wildtype optic vesicle into pax-6 neg ectoderm

no lens forms

in regards to pax-6 expression in mice lens formation, if you implant pax-6 optic vesicle into pax-6 neg ectoderm

no lens forms

How can 1 signal do so much?

the same inductive signal can be processed differently by diff cells depending on:

• the other signals a cell receives

• the state of the cell receiving the signal

Sequential inductions

• one way how complicated arrangements of multiple tissues can form

  • when cells closest to an induced signal respond to the signal and change, then induce the next cell to do the same, and domino affect goes on and on

  • so how one cell inducts the next, which inducts the next

Morphogens

• one way complicated arrangements of multiple tissues can form

  • morphogens work by the same signal inducing diff outcomes based on the concentration of morphogen received by a certain cell (so based on how far the cell is from the morphogen secreting cell)

  • ex: BMP4 is NOT a morphogen to induce lens development in the eye (if over a certain threshold, a lens is formed)

  • high BMP4 gives certain forms of ectoderm vs neural tissue (will be seen later) so BMP4 can be a morphogen for other things

some signals are morphogens in one context but

not in others

• ex: BMP4

how to maintain a cell’s differentiation

cell memory

• can be accomplished via:

  • feedback loops

  • DNA methylation

  • changes in chromatin state

autologous feedback loops to maintain cell memory

• work within an individual cell where a signal initiates a signaling pathway that turns on the transcription of a TF necessary for that change in behavior

• then that TF regulates expression of its own gene, so you no longer require that initial signal

OR

• signal leads to pathway that causes transcription of a protein that is the signal itself, so the cell remembers what it is supposed to do

• both are autologous bc they work on themself

paracrine feedback loops to maintain cell memory

• between 2 adjacent cells

• occurs at the border between 2 tissue types

• one cell makes a signal that the next by cell uses to signal it to make a protein, which signals the first cell back

• so cells can communicate that they are different and say “I’m right here”

maintaining cell memory via epigenetic mechanisms

• altering chromatin state to lock in cell differentiation by methylated regions of DNA you don’t want to use and acetylate regions you do want to use

• this is why reversing cell differentiation is so hard, bc its hard to get rid of methylation and acetylation patterns

maintaining cell memory via changing TADs to reinforce gene expression patterns

• usually the last thing used to maintain cell memory

• helps keep a certain gene expressed or not expressed