[Music]

so today we will focus on transcription

factor and then we will move on to our

modifications to DNA itself and how they

influence differential gene expression

and therefore development okay so

transcription factors usually contain

three roughly you know three different

domains this doesn't mean all of them

have all the three all the time so but

by and large most of them have this a

DNA binding domain where actually this

is part of the protein that binds to the

DNA directly in amino acids interacting

with the DNA double helix

I'm the transactive ating domain where

other proteins or factors that bind

might modulate the activity of that

particular trans a transcription factor

and a third one protein protein

interaction so where or either some of

the transcription factors for example

the example that we are going to see

acts as a dimer so the two polypeptide

chains interact in that region

sometimes other proteins interact and

influence their activity okay so the

translating one is the one that's

responsible for actually activating or

not activating the RNA pol - eventually

so and these are you know that their

effect is really dramatic and therefore

we are going to see an real example

where the defect causes a disease just

to keep you you know motivated to

listening to this

an example is ma TF so this

transcription factor is expressed in ear

skin and pigment forming cells of the I

know tree like irises and if you have

mutation you'll have problem hearing and

multicolor irises and then wait for luck

as you see in this picture see the

mother has wait for luck and her

daughter Anza guess

genetically inherited so very specific

problems that you have when you have a

specific transcription factor missing so

their activity is very important and so

this gives you going back to those three

domains so in this protein dimerizes so

the middle portion or you know where

it's marked where you have protein

protein interaction domains so here that

helps them to dimerize and this long

carboxy terminal region is the one that

helps in recruiting other proteins you

know for example a histone deacetylase

etc and the amino terminal region is

where the DNA binding domain is located

so this DNA binding domain there are

different types of DNA binding domains

and based on that the transcription

factors are classified into several

classes and within those classes small

variations in the sequence might define

which promoter they bind which promoter

they don't bind to give you an idea we

look at some of them as a table which is

there in the book like home your domain

so that is a particular DNA binding

domain that is conserved and that is

present in you know these proteins

listed here okay so we will see them the

Hox protein in detail several lectures

later from now and then some have this

helix-loop-helix so HLH and that is

present in these transcription factors

and these are their functions listed

here in the rightmost column and then

losing zipper they help in you know

binding to a forum like zipper like

structure based on the leucine presence

it's usually every seventh amino acid

will be losing in them and then you have

these zinc finger motifs these were

historically described discovered much

earlier than others so these coordinated

zinc okay and that helps in interacting

with the DNA and this is again present

in these proteins is crippled in greld

these are all originally discovered in

drosophila and the

names are based on the mutant phenotype

okay and they are expressed in these

tissues a nuclear hormone receptors they

have zinc finger and they are present in

the steroid hormone receptors and then

sry socks that's another domain so these

are the classes based on variations in

the DNA binding domain structure okay so

how do they function like how do they

activate or inactivate or control

transcription oftentimes it's by one of

these two are both when they recruit is

stone modifying enzymes you know for

example when a transcription factor

binds to a particular sequence then this

transcription factor may recruit you

know DNA sorry histone acetylase okay or

they may recruit a DNA you know an

enzyme that removes methyl groups

inhibiting methyl groups from histones

and by doing that they will displace the

nucleosome structure and that DNA gets

opened up and more accessible for RNA

pol ii and other transcription factors

so essentially by altering the

modifications on histones they open of

the chromatin

okay so the Hallows transcription and

the second is the stabilized RNA pol ii

oftentimes RNA pol ii bound to the core

transcription factors as shown in this

cartoon it's not very stable but when

these transcription factors bound to

enhancer when they interact with all

these proteins they make a much more

stable initiation complex ok this they

stabilize RNA pol torn promoter

increasing the probability that that

will continue to elongate the initiate

the elongation phase okay

so in this structure you see the

enhancers can be at a great distance but

through protein protein interactions

that DNA can loop like this okay so so

the this explains Evie Dee's press

within the coding sequence also you know

in the introns or whatever or it can be

even the downstream sequence so these

are the general ways but there are

variations for each transcription factor

but this is generically if you look at

it these are the major ways by which

transcription factors help in

controlling the rate of transcription

alright so how powerful are they so here

you are seeing you know how the

digestive enzyme producing part of

pancreas called the exocrine cells you

know they usually produce the digestive

enzymes proteolytic enzymes and so on

and they do not produce the hormones

what hormones are produced by pancreas

anyone insulin glucagon anything else

okay to our garena insulin and glucagon

right these cells do not produce so here

this blue is simply showing the presence

of DNA in the nucleus

okay now you express three different

transcription factors in this okay PDA

x1 so this is expressed in the

pancreatic lineage starting from the

cells that originally required for the

intestinal tube formation in those cells

if some cells Express PD x1 they set the

pancreatic lineage and in there if you

have these two transcription factors

they become the endocrine cells of the

pancreas now here you are taken exocrine

cells this is in an organism okay it is

not in the in-vitro cell culture so this

is in the organism where in the in yo

Lian when you express these 3

transcription factors you have insulin

producing cells there so the insulin is

stained here with the red color and one

of these transcription factors is fused

to gfp so therefore you see green and

wherever both are there you get yellow

there in this you can see here you can

see and here

and so on okay so they are so powerful

they can change the fate of a cell okay

from EXO crying fate to endocrine fate

so they are very powerful so of course

now more dramatic things have been done

okay so if people have shown by

expressing a few transcription factors

any differentiated cell can be actually

converted into undifferentiated

pluripotent cells so that is even more

dramatic than this so this leads to a

few questions okay how do transcription

factors themselves get expressed in a

tissue specific manner okay so that's

the question so the answer is quite

simple like the stories that people tell

you know when I was a kid I had one know

one person who was several years older

to me like I when I was in elementary

school this person was in college so I

he he talked about some game that he

plays and I asked who taught you this he

said he is but you know what Petey

master who taught him then he's PD

master so I kept on asking and I never

got the relevant answers because the

relevant ions there is someone first

time discovered it so similarly why is

this transcription factor expressed in

endocrine cells because another

transcription factor activated it what

is the heart active only in pancreatic

lineage it's because another one

activated it in the endocrine the

endoderm lineage so that lead system

what is called transcription factor

cascades okay so they work in Cascades

example mbx activates pack six pack six

activates we saw already crystalline you

know in the lanes and in saline blue go

on summit of studying this another

pancreatic hormone okay so etc and

similarly my OD this muscle specific

really powerful transcription factor you

know that activates my Oh Janine which

activates other genes involved in

skeletal muscle differentiation so it's

so on and so forth you know like one

after the other so so the main concept

here is there is a cascade okay then if

you follow up the Cascade

all the way all the way up to the top

then you have something called Pioneer

transcription factors these

transcription factors can really open up

a highly condensed heterochromatin and

initiate transcription okay so it may

not be already poised for access to

proteins a good example is this PBX okay

so it can go and bind to sequences in a

highly condensed repressed chromatin so

that's the definition for pioneer

transcription factors and it probably

binds to inhibitors bound to that

repressed chromatin but then once this

transcription factor binds it can

recruit like for example my OD

transcription factor and it will come

with other accessory factors which help

in really activating the transcription

finally and like open up the place so

that this MEF 3mf 2 etc can come and

bind to their respective enhancers and

initiate transcription so these are the

Pioneer transcription factors and on top

of it you have proteins like the

Drosophila polycomb-group complex

protein and try thorax you need not

write down it's there in the book so

these proteins are bind to the histone

modifications and maintain a memory of

this original activation okay

memory of meaning when that particular

cell whose fate is specified when that

is going to divide within tired

individual organism okay during

antigenic stage um all the cell's

descendants of that particular cell they

will all know that I have to keep this

region active this region suppressed so

those are done by those proteins the

polycomb and tri thorax group proteins

so for now you don't need to worry we

will see that concept with the DNA

modification also in in couple of slides

so so this is all about transacting

factors controlling transcription so now

there is an opposite phenomena also in

hand

like that there are other DNA sequences

that act as negative enhancers meaning

their sequence prevents the spread of an

activation activity like for example if

an enhancer activates and if it is going

to disassemble the nuclei in nucleosome

so now is that going to spread along the

length of the chromosome and adjacent

genes will get activated or what so you

don't want that right you want that

particular factor to be expressed in

that tissue not all genes so something

has to restrict that and further you

have DNA sequences to which proteins

bind which insulate or restrict these

enhancer activities okay so that is what

we are going to see next and they are

often called silencers okay silencers so

silencers are like opposite of than

enhancers so so here is one example so

here you have a gene called this not

gene fi an element called neural

restrictive silencer element so what it

does is it binds proteins that protein

is expressed in all tissues except in

neurons

so as a result in all the tissues this

sequence will be bound by the protein

that binds to this and there the the

genes that are under the influence of

this particular enhancer will not be

expressed

so therefore the genes downstream of

those promoters will be expressed only

in neurons and as a result this is

called neural restrictive silence so

here is a reporter where instead of the

actual gene you have lags that because

you can assay the lags at encoded

proteins activity so when you have this

silencer sequence or just send to it now

you find it's expressed only in the

central nervous system here the embryo

11.5 Dave old mouse embryo and if you do

not have that silencer element it's

expressed everywhere

so so these do opposite of enhancers

they restrict the influence otherwise

what will happen is the enhancer effect

will not be very specific and restricted

to the genes of genes that need to be

activated it will spread and the control

will not be really a tight control you

know adjacent genes may be partially

activated etc so the next we go to

modifications that happen to the DNA

itself okay so initially we saw that do

not get confused here okay so we already

saw methylation etc and that was at the

histone proteins and that affects the

chromatin structure architecture okay

whether it is tightly coiled with

nucleosomes and histone h1 that is

bringing all those nucleosomes together

into that a solenoid structure or it is

going to be opened up okay methylation

distillation all that we saw and there

we saw some methylation sin h3 tail can

actually be activating right so do not

forget that because oftentimes you may

be mislead by that you will ah just

automatically assume methylation means

inactivation be in our seat elation

means activation acetylation that is

true that generalization is true but not

for methylation so now we are going to

look at methylation that happen to DNA

so so I told you to perpetuate an active

state or repressed state we have those

try thorax and polycomb proteins that

bind to the you know the modified

histones like for example if something

is acetylated and you want that to be

active these products proteins bind

there and they maintain that state of

active activation activated state um but

very similarly but more robust is the

modifications that happen to the DNA

okay and that modification happens by

methylating the cytosine residues okay

so you can have a ch3 added

therefore you are the fifth base

5-methylcytosine okay so this matters a

lot in regulation

so here methylation usually means

repressed state like inactive that gene

is not going to be transcribed and this

can be perpetuated through mitotic cell

divisions okay we will see that how that

happens in couple of slides we will see

that and the second this can have

developmental time factor involved in it

okay like modification happens at some

space and time not all the time okay so

a good example of that is shown by the

way hemoglobin genes are expressed the

beta globin if you take how it is

expressed in that you can see a good

example initially in the early embryo

you have this epsilon version of the

globin gene is expressed its promoter is

not methylated okay whereas at that time

point you have the gamma globin which is

usually expressed in the fetus that is

methylated and it is not expressed and

later as embryo progresses then epsilon

gets D methylated and that becomes

active this gene gets turned on okay it

was dormant and now it is turned on well

this epsilon globin gene gets turned off

and when the day you know infant starts

to grow what happens is even this gamma

globin gets methylated and inactivated

and beta globin gene gets activated and

that is what is expressed in our body so

in all of our gene of you have the

sequence epsilon and gamma sequence but

they are methylated and not expressed

they were expressed sequence ly during

your embryonic and childhood development

so now you have only beta globin

activation and these have consequences

okay if there is a problem with this you

may have heard this deceased Alice Samia

that results from failure in the

sequential methylation demethylation so

here what happens is in these patients

or you may have problem activating the

beta-globin let us say you have a

mutation in beta globin and now you do

not have a functional globin protein

produced although you have perfectly

good copies of the gene in the

chromosome but unfortunately they are

methylated so the methylation program

has not been affected they are active

and methylated but the gene is not

expressed although you have wild type

copy and that's how you get this beta

thalassemia Manolis beta globin gene

that's involved so this is a very well

characterized congenital disease seen in

India particularly or you know some

pockets bordering on Iran thermal order

that area in certain communities of

communities where you have marriage

among close relatives like first cousin

marriage or sometimes an uncle marrying

a nice you know elder sister's daughter

that sisters brother married you know

those are not uncommon maybe they are

uncommon now but couple of generations

ago they were not uncommon in those

families what happens is like for

example this given person sister may be

heterozygous and this guy also may be

heterozygous because they come from the

same parent and they survived because

they are heterozygous now there is one

fourth chance their child will be

homozygous for the mutant allele so that

is how you have beta thalassemia running

in families okay and the underlying

causes this methylation for issues so

now how do you perpetuate this so that

is where we are getting to so usually

these methylation block transcription by

preventing transcription factors binding

to enhancer sometimes it is the opposite

as well

like some inhibitors will bind it to the

unmethylated one and they will not bind

methylated okay in this particular

quarter and what we have is this

sequence okay so you have CG coming

together so this is often called CPG

Islands and its significance will become

clear in couple of slides so for now do

not worry you just think that this

promoter region is usually subject to

methylation demethylation so when it is

not methylated a transcription factor

binds and activates transcription from

the downstream promoter okay and if it

is methylated this transcription factor

does not bind as a result the gene is

not active okay so therefore here this

example shows you that DNA methylation

blocks transcription factor binding to

an enhancer okay and another way by

which they function is this methylated

cytosine may recruit a protein like in

this case mecp2 which can do two things

one remove the acetylation mark by

recruiting a histone deacetylase and

recruit a histone methyltransferase and

the mark you know histones with

inhibitory methyl groups okay and due to

these two actions on these methylated

promoters end up blocking transcription

this sort of methylation based

transcriptional repression can be

perpetuated through mitosis simply

because they usually these cytosine are

always adjacent to a guanosine residue

CPG Island the phosphate in between

probably helps in pronouncing better

otherwise I would say CG so CG repeats

normally people call CPG repeats CPG

Islands meaning that they in the

chromosome here and there you have a lot

of repeats of CPG and that's called CPG

Island and these are recognized by a

methyl transferase called dnmt 3 this

does not

need either one of the two seas that you

see here CCG means the opposite strand

will be GC right so you have C in both

the strands due to this base

complementarity CG means in the opposite

for that G does vs C so here neither C's

are methylated and such sequences can be

recognized by this methyl transferase 3

and that's why it's called de novo

methyl transferase it can start from no

prior information for that and it

methylates that now you have a

perpetuating methyl transferase that you

need to remember this methyl group is

not erased during mitosis it's going to

remain remain there now

after replication one strand will have

the cytosine methylated the other one

will not have and this one recognizes

such methylated cytosines

and they methylated in the opposite

strand the nearest see and third is of

the adjacent g becomes crucial for this

so now it gets methylated so now both

strands are methylated now you undergo

DNA replication then one strand will be

methylated another one will not be and

this D and Mt will empty one will do the

methylation of that so this is how this

representation be maintained during cell

divisions so only on in the embryo

embryonic development at some point you

inactivated by methylation let us say

the transcription factor cascade and you

chromosome modifying they ended up

methylating the DNA now all the cell's

descending from that of original cell

they will all maintain that active or

inactive state okay so that is how this

works and so this has a lot of

significant consequences for you know

many situations particularly here if we

look at this dosage compensation okay so

what is dosage compensation so like for

example in mammals like human if you

take females have two X chromosomes

males

only one X chromosome well the y

chromosome doesn't have many really

vital genes but the X chromosome has a

lot of important genes okay so now will

the females have one extra load like

will the protein produce from them be

double that of males and build that not

cause a problem in terms of the

phenotype right so that has to be taken

care so the odd is usually taken care by

one of three different mechanisms like

for example if you take C elegans both

the X chromosomes get reduced by half

and therefore you have the final

quantities like one compared to the male

that will have only one x chromosome in

drosophila what happens is the male

single X chromosome is doubled up okay

its chromatin is modified such that the

nucleus it's it's truly you chromatin

and the output is more efficient and

therefore it is doubled up and in human

we do the opposite one of the two X

chromosomes in the female is totally

converted into heterochromatin and

repressed okay and that is what you are

seeing in this you know cell it's a

human derived cell very this arrow

points to a large black region and that

is the condensed inactive X chromosome

and this is from a person with 3 X

chromosomes and therefore you see two

black things these are called bar bodies

ok so that is how the United a chinois

--kx okay what's the big deal for me now

the big deal is if you look at the B and

C and if you look at B what do you have

is it's a very early embryo in this you

have the reporter lags at fused to the

promoter on met paternal X chromosome

okay so lags that will be expressed

if the paternal X chromosome is active

okay otherwise there will be no lags at

and therefore this blue color will not

happen so the pink cells are simply

where the paternal X chromosome is not

expressing it's not working so this is

very early you see most of the cells

having this color so this is the inner

cell mass from which the entire embryo

develops but when you go to the later

stage if you look at it here in these

cells you do not see the lags at

expression so it's turns out that you

know in Mouse the Trop of flash cells

preferentially inactivate the you know X

chromosome of paternal origin but in

other region you can clearly see both

kinds mixed okay and there are three

important points to remember about this

inactivation when the starts early in

the embryo okay meaning in your least

age in one cell if it is inactive and

then you have an entire tissue or a part

of a tissue derived from it all of that

will not have that gene expression okay

even is paternal paternal expression

will not be there if it is maternal if

maternal is inactivated then that will

not be there and second it is random in

which cell which x chromosome which

meaning I am referring to maternal or

paternal is going to be inactivated is

random and once then it is irreversible

it remains in all the descendants of

that lineage and due to that you can

have patches of variations now in the

somatic body and that is often very

readily visible in organisms where you

have skin color that is you know having

patches okay even see this in calico

cats they have that issue we saw that in

the somatic cloning x-ray experiment

and that is how those variations

developed okay so these three points you

need to remember it happens very early

and it can happen random and once

happens it is irreversible all the

descendants allows that inactivation so

if this is the case then if I have a

gene on X chromosome and that is very

vital and if that gets inactivated in my

father's genome let us say that means

having one wild type copy from my mother

is not worthy enough okay for certain

genes I need my mother's copy for sure

mother's allele is required and

similarly for some genes I for sure need

the father's copy so this is where you

will find that the you know when you are

drawing punnett square right it doesn't

matter where the allele comes from right

maternal or paternal doesn't matter but

there are situations where like this X

chromosome dosage compensation where

that matters so we will see that in the

next set of slides okay

whatever methylation pattern that exists

they get all of them get erased during

gamete or Genesis the new methylations

happen this does not happen to all genes

by the way there are certain genes that

are methylated and those genes are going

to be methylated in one way if the

gamete of Genesis is happening in a male

body happens in another way if it

happens in the female body

for example certain genes may be

methylated during spermatogenesis only

and certain other genes may be

methylated only during wood genesis and

usually they are mutually exclusive

genes that are methylated during

oogenesis are not methylated during

spermatogenesis and vice versa okay part

is what we call as genome imprinting so

simply what is genome imprinting six

specific methylation as a conscious

grunts sex-specific expression if that

particular gene is methylated during

spermatogenesis it's not going to be

expressed in the from the paternal

allele in the offspring if it is

methylated during wood genesis that

particular gene is not going to be

expressed from the maternal allele okay

so you have two alleles assume both are

wild-type it does not matter which one

is inactive as long as one is definitely

going to be inactive then dosage

compensation will not come into picture

right because one copy only you're going

to have so so the odd is how this works

and as a result you will not have both

alleles okay like for example you take

gene yay that is inactivated during

oogenesis now even if you have a

wild-type allele it is not going to be

useful so you have to have the paternal

allele because only paternal is not

methylated and as a result it's going to

be expressed from there and the same

logic holds in the opposite direction -

so the germline uses in a very different

set of combination of the existing

moniker biology rules in taking care of

its genome okay you can't readily mess

with its genome and the way it protects

has its own idiosyncrasies okay and one

of them is this erasing all the

methylation and the newly methylate and

the new methylation is going to be based

on whether you are a female or male okay

so you could have got that particular

base pair sequence could have come from

your mother now if you are male you will

have a male specific methylation added

to that gene which originally you came

from a maternal like a female source and

vice versa so the methylation is sex

specific sex of the individual in whom

the game it is mommy

now as a result for such genes je

imprinted genes imprinted meanings

expressing methylation it is subject to

for them you need to have both the

alleles because one allele is going to

be inactivated and the other is going to

be the only one available so if that is

the case if you have a mutation in such

genes then you will have sex specific

you know phenotypic outcome so that is

what we are going to see next so this is

a cartoon so the next will be an actual

disease so here if you look at it this I

G of 2 this is not transcribed so here

is a situation where there is no

methylation but a factor binds there

okay so this binds to unmethylated DNA

remember in the previous case like

several slides ago we saw a situation

where the activator by instant

unmethylated DNA and activator does not

bind to methylated so here what we are

finding is methylation in activates so

this IEF 2 will not be produced when

this is not methylated okay that is the

case in the maternal genome here this is

methylated in the paternal chromosome

like what is mean what it means is this

locus gets methylated during

spermatogenesis not during oogenesis now

you have an insulated protein binding so

these are the proteins that bind to

those rest silencers and that binds and

insulates this coding sequence from the

effect of the same answer so this just

happens to share the same enhancer so

vini not worry here so this is produced

but not this this restricts that but in

this form a derived chromosome you have

a methyl group there as the result this

insulator does not bind and this

enhancer activity imparts transcription

from igf2 and that gets expressed so you

need to have both copies if you

this mutated okay like the father carry

the mutant aversion then you will have

due to mutation you will not have IGF to

protein and your maternal although as a

wild type copy it is not expressed so

due to that you are going to be

deficient of this protein and that is it

hypothetical no it does have real-life

consequences so you take these two

syndromes you know Angela man syndrome

and prader-willi syndrome

okay these are two different phenotypes

you know coming from mental retardation

and caesars and so on you know both have

defects one is the Angelman syndrome is

more severe than the Prada really but

double is lethal so if you look at the

the first one in this punnett square

you have the chromosome 15 this is

coming let us say from a wild-type sperm

and a wild-type egg

you're totally wild-type now let us say

you have a particular region in 15 is

deleted in the mail so now you have a

wild-type chromos copy of that

chromosome chromosome 15 wild-type

allele is coming from the egg but that

is not helpful because the required

genes are actually inactivated due to

maternal specific in imprinting and when

you don't have that from the sperm then

you get this particular disease okay it

is because of the those particular genes

that normally gets expressed from the

maternal paternal copy now if you look

at the opposite where you have while

site chromosome coming from the male but

a deletion coming from the female a

different set of genes are affected

because the methylation is six specific

here and now the corresponding or

Lil's in the mail or inactivated and you

need it from the maternal copy and that

is not available due to the mutation and

due to the difference in the genes

affected you get a Angelman syndrome

okay so this locus is shown in the next

cartoon so there's a complex locus where

you have several you know genes there

the gray indicates inactivation except

here this is a type of okay or in the

drawing whatever error they hear in the

book so this is active in male so you

have due to methylation this particular

one activates all these genes from the

maternal chromosome but not from the pet

owners are a sorry from the paternal but

in the maternal due to this methylation

here you have a block of that and due to

this these genes are not expressed so

one set of genes for example in this

particular case this one is expressed

only from the maternal copy while all

these blue ones are expressed from the

paternal and when you need both the gene

products then you need to have both the

alleles ok maternal paternal both

alleles and this is why you get those

businesses so this is what is genome

imprinting which is a calm consequence

of six specific methylation the key you

need to remember is existing methylation

marks are erased during gamete or

Genesis alright so right now in your

cells you will have

imprinting ok the paternal allele will

know I am paternal allele because of a

paternal specific methylation maternal

allele will know I am from maternal

because of maternal specific methylation

or the lack of in both cases and when

both other it is fine now in your germ

cells when they are going to enter into

gamete or genesis these marks are erased

okay reset button hit and no methylation

now if it is spermatogenesis you are

going to methylate a certain

you know low side and ER if it is wood

Genesis then you are going to imprint

again start another low side these are

mutually exclusive so I'm stopping here

if you have questions feel free

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