[Music]

all right so yesterday we were you know

refreshing our memory about Hawaii you

care leave gene looks right different

parts now ok this lady's here if I got

away of blocking it I can immediately

ask a quiz and see whether you remember

so I really find the cap fire prime

anywhere in the video at the very

beginning of the UTI ok so what will be

the relationship of transcription start

site with the relation to the exons

introns no it can't be hey there ok so

the Phi prime UTR can very well be

starting at the transcription start site

right so it can be in the exon - alright

we'll do more quizzes later on this so

right now we will continue on this so

today what we are going to do is mostly

focusing on molecular biology techniques

some of which you may have learnt in

other courses and some of them may be

new because some of them are a whole

organism based techniques and that you

may not have heard elsewhere so

essentially what we are the main focus

is what are the techniques by which we

try to understand differential gene

expression ok so that is going to be the

focus for this lecture as well as the

next one so this is continuing on

refreshing our memory of the eukaryotic

gene structure so the previous one was a

cartoon this is an actual sequence ok of

the beta globin gene so you have the

upstream promoter elements like this

Tata box

Tata box here and this is the -70

sequence so these are there in most of

the promoters that's why they are part

of the core structure here so this is

the sequence that signals the cap

addition and you see this is right away

is an exon the colored ones are exons

the gray or the introns ok so so you see

that the utr is an exon and there you

have a curve start codon

then this the actual nucleotide and the

translated amino acid sequence then you

have the beginning of an intron and end

of an intron and then so on then at the

end you of the stop codon and any other

utr 3 prime UTR right so then you are

the AAT Triple A this is a canonical

poly a signal signal but there are other

variants of this that also work as

polyadenylation signal so they help in

identifying a cleavage site also so

actually the machinery that does the

cleavage what the cleavage here means is

the pre-major the pre mRNA nascent

transcript can be longer at the 3 prime

than the mature on in the cytoplasm

having the poly a tail so at the 3 prime

there is a cleavage that happens so some

amount of the transcribed mRNA is

removed and to the remainder you add the

poly a tail so dart itself is a major

complex and highly regulated it is as

big as a ribosome ok another such major

complex is this place or so okay the

splicing machinery so these are major

protein complexes that operate so so

next we are looking at transcription

itself so this the first step here so

you have the transcription so you have

as soon as the nascent mRNA comes out or

nuclear mr.and are a name so you have

the cap added by methyl G capped at that

stage splicing is not done yet okay

that's what I want to point out these

auditions help in protecting or a name

okay so the but I'm not sure what that

is the reason why that step is fast but

these auditions that both ends happen

before splicing so then you have

processing which is essentially splicing

then you are the messenger RNA or mature

mRNA so this comes into the cytoplasm

where it gets translated then you have

the protein chain

and then that I asked to undergo two

things one proper folding to the native

conformation and also post translational

modification so here for example this

prosthetic group it's added subunits of

you know this is a four subunit protein

it's a heterodimer so beta globin Alberg

globin you know to each come together to

make the functional molecule so all

these tips put together is what you call

as gene expression okay so do not assume

only transcription is gene expression

and this is something like protein level

the whole thing is gene expression when

you talk gene the definition of genus

biological function that is the

phenotype so protein is obviously

responsible for it so we do not

distinguish these steps all the steps

from the chromatin structure

modifications to the chromatin structure

like from heterochromatin to you

chromatin transformation that happens

due to removal of methyl groups and

addition of acetyl groups and specific

methyl groups to h3 tail starting from

there to the post translationally

modified active or inactive protein so

up to that the whole steps are called

gene expression each one is one step in

gene expression so now we know what is a

gene and what is its final functional

form so now let us look at again we are

refreshing some other steps we are still

not getting into differential gene

expression for that we want to

familiarize with what happens so that we

will know where regulations can happen

so the first thing is we are going to

look at the transcription initiation

itself so transcription initiation to

this cotton goes with some details but

the summary is that a set of proteins

have to bind in a certain sequence to

enable the RNA polymerase to to start

transcription so the initial assembly

and recruiting the RNA polymerase to the

promoter is what you call as

transcription initiation so here you

have TF

Toodee transcription factor 2d so that

has to first bind it binds the tata box

then recruits to e8 2d sorry - yeah and

then you have B and H so these clamp

like structures the add and then only

you have Paul to recruited and to the

Paul - you have the e and EF already

binding to it and when it binds this

carboxy terminal domain CTD plays a

major role in regulation so dart is

bound to the 2d structure this main

molecule and Sedaris Avedis so this is

already set but it is not go okay so

that requires phosphorylation of CTD so

- phosphor lists are not to

phosphorylate each step there if you

don't phosphorylate no transcription

although everything is bound and it is

already so the odd is a step where

regulation often happens so once this

gets phosphorylated you know there are

certain serine residues that are

critical for the phosphorylation so

oftentimes developmental biologists use

those phosphorylated serine specific

antibodies Ct phosphorylated CTD

specific antibodies to measure global

level of whether transcription is

happening in a given nucleus or not okay

so with those phospho specific

antibodies CTD specific for you know

phosphorylated CTD specific antibodies

if you do not detect a signal probably

there is no mRNA transcription happening

so remember we are talking only mRNA

buddies all it is because it's Paul to

specific antibody we are talking about

so other transcription could be

happening so once it is phosphorylated

then it is released from TF to D and it

is ready to elongate so initiation then

elongation okay so this is initiation

step and this is elongation and even in

a elongation it can be passed sometimes

okay so those details we are not going

to get into but this is good enough to

know that this is a potential step for

regulation alright so now to be done

with those basics so we are going to now

look at a series of experiments that

help us to identify sequences in the

promoter region and as well as elsewhere

in the vicinity of gene that could be

contributing to translation sorry

transcription 1 and second we are also

going to look at how does one find

transcription factors that is

transacting factors like proteins ok the

DNA sequence itself we are going to say

sis because it is in the same molecule

as the open reading frame and protein is

encoded elsewhere and it comes as a

different molecule different from that

piece of DNA value of the gene so

therefore it is called trans so when you

say transacting factors you are talking

about factors other than a given gene

sequence that influences the gene

expression okay so so this is a very

commonly used as a people use it for

multiple contexts where you want to test

nucleic acid protein interaction ok so

right now we are looking at DNA protein

interaction to find out the promoter

elements that may be involved in

interacting with a specific

transcription factor so these

transcription factors essentially like

you already saw a base so these

molecules thus shown in this blue color

so these are aiding in recruiting the

RNA pol toe to the promoter and they are

transcription factors so these factors

listed here and named these are there

for every gene these are core

transcription factors and there are gene

specific transcription factors that we

will learn later and so how do we test

whether a given such transacting factor

interacts with a given element of DNA or

not so to test that is what we use this

experiment called a gel shift assay or

gel mobility shift assay or gel

retardation assay

there are multiple names for it most

commonly used to term is electro

phoretic mobility shift assay or EMS AR

EMSA okay so some labs columns or some

labs called EMS a some labs do not even

say that they say mobility shift gel

shift okay it's extremely simple

technique so what you are actually doing

is you take a radio labeled aversion

shown here in this you know whatever

color that comes on that screen here if

for me it looks purple so you take radio

labeled aversion of the DNA fragment you

want to test whether it interacts with a

given protein then you incubate with you

know the protein it is it may be either

purified protein or it's a cell lysate

where the protein may be present so you

incubate with it

and then you run on a gel if the protein

binds to that DNA fragment then the

complex size is bigger than the

individual nucleic acid so the mobility

is reduced since in the gel you see it

as a shift you know an upward shift you

call it as gel mobility shift assay or

since it retards the mobility of the

nucleic acid you call it as gel

retardation assay so all those names are

really valid names so so that is how you

see it so here in this particular case

pack six is a transcription factor about

which we will see some more and without

that the free nucleic acid we call it as

free probe so free probe for a given

time in a given conditions of gel for

example gel pore size etc or it moves a

certain distance and when the

transcription factor is added which

binds this particular DNA fragment its

mobility is shifted since it's radio

labeled you can detect it by

autoradiography okay so this is a very

versatile and very useful experiment in

our lab we primarily used it for by

finding RNA binding proteins that

interact with three prime UTR and reveal

translation so that's the context in

which we have used so therefore it is

useful for nucleic acid protein

interaction in different contexts so in

three you have park six added so it

shifts all right

and in lane 4 also Park six is added

it's a control experiment so here what

you are doing is when yeah it binds then

there is there are two alternative

explanations possible maybe it is a

protein that would bind any nucleic acid

okay non-specifically it may not be

sequence specific to test that what do

you do is you add a large mole or excess

of the same nucleic acid fragment but

without radio labeling okay so now what

will happen depending on what is the

molar excess let us say I have tenfold

more Rx's or 100 fold molar excess then

this intensity will go down here there

is only one because it's a cartoon and

just showing you the you know the scheme

but in an actual experiment we have to

fold fourfold ten fold hundred fold like

that serially will be increasing the

unlabeled the same nucleic acid so that

would also have equal tendency to bind

it it's more like a competitive

inhibitor so now you have the band there

but that is not having radioactivity so

you don't detect it okay so that and

then another control which is not shown

here is you add similar mole or excess

of similar number of nucleotides

okay similar length of the nucleic acid

but of a nonspecific sequence and that

will not compete out if it is

sequence-specific binding same sequence

unlabeled will compete out the radio

label there but a different sequence

will not compete so even in its excess

this protein will still bind it to the

that particular sequence alright so the

next one is DNA's protection RC so this

you do it see this is looking sort of

not familiar from our facial expression

I can detect the head that is because

you are no longer doing DNA sequencing

by this method but otherwise suppose

like when I was a student this is a

routine gel so this kind of image you

see every day if you are doing DNA

sequencing so on those days we didn't

really do whole lot in developmental

biology so these molecular biology

experiments is what most of the people

did so this is how you do DNA sequencing

so you have radio labeled all eager to

start with and in each reaction you have

dye deoxy of that nucleotide so you have

four lanes you will run four lanes on a

gel and then you look at in which lane

you have the smallest fragment and from

there progressively you count up four

and that's how you get the DNA

sequencing so this is one such DNA

sequencing gel but here you are not

sequencing the DNA itself so instead

what you are doing is the DNA is

incubated with a protein that binds to

some region so this is really large

fragment compared to what is used in gel

shift assay okay so here you are

actually trying to find which sequence

in a larger sequence binds to the

protein then once the protein binds to

the DNA you add a nucleus a controlled a

nucleus treatment you do so now what

happens is the fragment that is

protected by the first protein that

bound to it protects the that's fresh

sequence and the rest of it gets cleaved

and when you run a gel since it is a

controlled digestion and your partial

digestion for every you know nucleotide

length so in the control Lane you see

bands for the entire length because at

every place that could have been

digestion but if you look at this

portion in these two lanes and then here

you see bands missing when you have a

DAT pack six okay so if you compare this

with this as well as this region in the

control with this so indicating that

pack six binds to that region

essentially you're testing whether your

protein protects which portion of a DNA

fragment does it protect from a nucleus

cleavage so this is also done with the

RNAs protection also for to find you

know part of the RNA wear and RNA

meaning protein binds so this is DNA's

protection I said this is also called

footprint assay okay because it's like a

footprint the proteins footprint so now

before we go into next set of techniques

we are going to look at a part of the

gene okay it's a regulatory element it's

not only really that coding part and it

can be anywhere actually it need not be

always in the promoter region it can be

a considerable distance it can be

downstream or of the transcription

initiation side as well so that is what

we are going to look at and they are

called enhancers so what are enhancers

the first point tells you they control

efficiency and rate of transcription

okay so they may increase the rate or in

some places they very strictly specify

spatial or temporal expression of a gene

okay so there is shown here in this this

is the pack six promoter cartoon so

these orange colored bars are bands or

the exons

okay this red one is the promoter itself

then this green is the enhancer and you

see enhancer present here in one two

three four fragments for parts and one

of them is actually in an intron okay

this is an exon and this one exon and

this whole thing is introns it's part of

an intron - ok so all these enhancers as

I told you they you know control

efficiency and rate and sometimes very

strict temporal and spatial regulation

and here we see such a spatial

regulation information for example this

enhancer between exon four and five

this confers expression of this gene

that is pack six in retina okay and this

particular exon here just before the

transcription start is a neural tube in

answer that make sure this protein is

expressed in neural tubes and the other

one lens and cornea if you don't have it

it's not gonna express there and the

other one that upstream most pancreas

specific so each one of these four have

four different tissue specific

expression of this gene so you may not

have the core promoter code

transcription factor everything but you

are not going to have transcription if

you do not have these enhancers and then

those enhancer specific transcription

factors okay so we will see them one by

one so this one this actual sequence

tells you in a part of this pancreatic

enhancer sequence where you see binding

sites for two different transcription

factors okay to try and two different

transcription factors bind to that so a

little bit more detail on that we will

see you know in two slides later okay

there are many ways by which one

identify same answers and here just one

easy to I didn't understand and very

powerful historically had been very

useful technique called enhancer trap so

what you are actually doing is you are

having a core promoter driving a

reporter gene all of it embedded in a

trans possible element okay so this is

useful in an organism where in natural

try endogenous transportable elements

they exist and they hop on and hop out

depending on the context you know where

the transpersonal element is active or

not depending on that they will move

around the genome and usually transpose

elements to get inserted or they need

repetitive sequences and wherever that

is there they will go so now if you use

the transpose ability

as a vehicle to carry this reporter with

the core promotor meaning no

transcription factor binding accessory

sequences like no enhancers or anything

and now you provide the context in which

transversal elements are active and then

you stop it and then look at all the

different insertions kind of mutagenesis

using trans possible elements okay

instead of some other mutagen here you

are using transversal elements so in

different individual there of that

organism you will have trans possible

element inserted in one part of the

genome or another part of the genome so

now suppose this transcription is this

transcriptional reporter gets inserted

in the vicinity of an enhancer as you

see in this picture here or like here it

has come next to an enhancer okay and

this enhancer normally controls the

expression of a gene downstream so this

is the gene here and now this enhancer

influences the expression of this then

this gets expressed where this gene is

normally expressed and then you can

detect it this way okay this is a

drosophila embryo so where you see the

expression pattern

so essentially you are trapping an

enhancer with the help of a reporter

gene so the reporter gene has a weak

promoter it just has the core basic one

so it's not going to express on its own

without coming under influence of an

answer so when you do random insertion

if it gets inserted near an enhancer

then it expresses where that in hands

are normally activates transcription and

that is how you find oh this same answer

works in this tissue so later that can

be verified by other methods as well

reporter gene is lags at here so this is

done in an era long before GFP was

discovered okay so we yeah odd you will

find out by sequencing the flanking

region so that will not be difficult but

the point is you have trapped an

enhancer okay so the primary goal again

these are all coming from the thought

process of genetics okay in Jena

you do not worry about the molecule

first you first find out did I hit a

molecule that is responsible for a

function then you can search and find

the molecule that's lot easier then

having a bucketful of molecules like you

know you can take any organism and crush

it and send it to a company and they

will give you all the proteins produced

there all the mRNA produced there but

what will you do with it without

connecting to the function okay so

normally we call that you get a big

laundry list but you don't know what to

do with it there are many such lists

that are hanging around for the last 20

years without people having made

progress this is not a belittle the

usefulness of that this is only to

highlight the importance of directly

connecting to the phenotype because it's

very easy to claw do an inverse PCR and

sequence than a neighboring sequence

because you know this sequence okay so

you can digest with restriction enzymes

and some might get some scut somewhere

here and there and under a very dilute

condition if you like a tit intra

molecular ligation will happen so it

will get circularized then you take the

sequence you know then you use primers

going in the opposite direction then

they will sequence the flanking rate PCR

amplify the flanking region then you

sequence and you will know verities okay

so that is how you identify but the

primary goal here is to find will I get

a tissue specific activation so that

means I am in the vicinity of any

potential enhancer so that's the goal of

this so these were done in the mid

eighties to late 80s period so the next

one is the same thing but helping you to

determine like for example if you know

an enhancer and then you you want to

find out what are all the different

places where that enhancer works so you

can make reporters under the control of

enhancer and look at it so this is an

older assay method where it is lags at

and then you see its expression for

primarily in you know central nervous

system here and muscle specific

expression and here what you are seeing

is

lenz crystallins announcer and you see

where it is expressed so this is images

I particularly like because it shows you

this is specific control so clearly

right so there are a lot of such things

you know yesterday in the lab we had

some images where the gene is a GFP is

expressed only in the new clay of a

particular set of cells along the length

of the warm okay so the Wang has

something called the same basically

horizontal structure providing

structural reinforcement for the body

and that is made up of 16 pairs of cells

and this particular gene is expressed

only in those 16 cells the rest of the

959 cells they are all dark and only

these glow green okay so it was too late

so I didn't make a picture that could be

shown here today so but this is doing

the same job so you see it's only in the

lens not even in the rest of the eye so

because you don't want crystalline

expressed elsewhere you want only in the

lens so now you are getting an idea of

what is differential gene expression

right all right no okay so for studying

the enhancers now what we are doing is

can I use the enhancer to do something

else you know applications of an

enhancer so they are very useful to two

different contexts we are going to see

two different examples one of them is

often used in Mouse where you want to

have conditional knockout let us say a

gene as a crucial role in cleavage stage

of the embryo and if you just hit it

okay null allele you create then it's

going to be embryonic lethal imagine

that gene had a function in the liver

and you want to know specifically what

it does in the liver you will never know

that

so therefore when people want to find

tissue specific or organ specific

function of a gene they want to have

conditional knockouts okay meaning

conditional deletion so this knockout at

all slang that is being used so widely

that it is also there in textbooks and

we use them because we understand the

meaning but other way basically what do

you mean it's deletion of a gene that's

what the non geneticists initially

called a knock out and now geneticists

also use it then some people call knock

in and then people say knock down okay

a partial depletion they call knock down

I just don't like that word because that

doesn't convey the meaning it doesn't

say what depletion tells and similarly

knock in meaning transgene and insertion

they call knock in okay so now we are

looking at conditional knockout meaning

we are not deleting it at the zygotic

nucleus so it is not going to be there

in all cells we are going to

specifically knock out in a particular

somatic tissue so how do you do that how

enhancers help so now you have one

strain of mouse okay where you have a

battery of Forge recombinase so okay I

tell you a little bit about the

bacteriophage I'm not sure how many of

you are loaned bacteriophage lifecycle

did anyone learn lamda fudge lysogeny

and like take cycles you know them so

therefore what about a battery of fudge

do for their living what do they do how

do they survive horses like bacteria is

the horse so they get integrated okay

and then when favourable conditions come

okay from that it's all they can make

multiple copies so they can come out

also so they can insert them and come

out how do they do they do it using

site-specific recombination and that is

what is being exploited here because if

I say recombinase and if you didn't know

reward is recombinase then there is no

point in going forward so Crees one such

recombinase okay so you express that

protein under the control in this

particular example enhancer for all the

mean okay so when you have a mouse train

that has created combination

under alba mean enhancer as a transgene

so now what this is going to do is it's

not going to express anywhere except in

the tissue in this particular case liver

where all the mean will be produced only

there Creary combination will be

produced now you have another mouth

strain where your gene of interest for

example in this case this transcription

vector hnf all for all for its exon to

is flanked by the sequence recognized by

the critic companies okay so that is how

the site specific recombination works

right so the site for the Kree is called

this is bacteria for p1 and that's

called loxp sites so the loxp site

flanks exon 2 so normally people call it

as fluxed okay flank debate lakhs of

fluxed so you flux the gene of interest

this excellent who is not going to be

removed in that strain anywhere because

there is no recombination ormally in the

mouse that will recognize lakhs piece so

that is going to be healthy and simply

expressing Cree recombinase in the liver

is not going to cause any problem for

that strain because it will work only if

there is a lock speak otherwise you are

producing a protein that's neither toxic

nor having any functional interference

but if you cross these two mice and

generate double mutant Mouse that

carries both of them in its liver you

will have both the functional elements

brought together you will have Creary

Khamenei's produced because all the mini

enhancer makes it expressed in the liver

nowhere else and you need to remember

this is germline flock sing so this is a

line it's a mouse strain so it is going

to be there this is the the sequence is

there in the genome of all the cells we

already know genome equivalence and that

doesn't matter

only in liver where you have the Kree

recombinase the loxp sites will be

recognized and it will treat in between

bacteriophage genome and it lakes ice

out and then you will not have axon - so

this is conditional knockout so this is

how people use enhancers applications of

enhancer

okay one of them so now in a joy in a

journal club or in a seminar if someone

says conditional knockout and I flux to

this game you immediately understand

what it is right so it's very important

to know this and second one this is

primarily used in Drosophila but it's

extremely powerful and they came up with

this for a long time ago and only

recently people have been successful

doing it in C elegans probably when

people did not find really important

context where they needed it or another

one lazy to develop a tool or whatever

but recently they have shown it works

but in Drosophila this has been very

well exploited and you know this this

one example is good enough to know how

powerful this is so this is gal for you

a a system very similar in you know in

principle to the previous one you will

have two different strains okay here one

strain under the enhancer of you know

this a particularly marginal disc okay I

don't know how many of you know imaginal

disc so imaginal disc goes back to

metamorphosis okay so drosophila being

any insect it is a warm stage right

caterpillar sort of stage from this the

fly comes out so there is metamorphosis

so in that warm like stage it has the

primordium for all the adult stages and

these primordia are what we call as

imaginal discs so you say ving primordia

means this wing imaginal disc means the

primordial cells that are present in the

caterpillar which will eventually be

expanded into being in the adult so

those are the imaginal discs so in a

very specific imaginal discs rather an

imaginal discs specific enhancer you use

that to control the expression of an

East transcription factor

for so gal4 again has a very specific

short sequence that it recognizes and

activates transcription so now you have

that transcription factor produced in

one strain of drosophila under this

imaginal disc specific enhancer now you

have another strain where you have the

in the upstream of your gene of interest

okay or the gene itself is a trans gene

for example pack six you are taking from

the mammalian system and putting it here

so you try to express this under the

control of the promoter where gal4 binds

okay so this is called the upstream

activating sequence that for you a a yes

so the us here is gal for us and that is

four pack six and in the other one you

have let us say we you know ie specific

email I imaginal disc okay so now when

you cross both and make a double mutant

you are going to produce pack six

protein where normally this will drive

the gal for expression so in this

particular case it is not I it is

another structure in the thorax or in

the head region where by driving pack

six they were able to induce the

formation of I there okay please

probably Anton or in this case I think I

already checked the book to see instead

of developing antennae you develop I so

what it tells you here is pack six acts

like a master regulator of eye

development so once you provide pack six

then looks like everything else is

already there in that particular tissue

to drive the formation of I so we will

see similar such a complete organ

formation in a wrong place or more

examples later as we go through a good

group of genes of the Hox genes or the

homeotic mutations

where you have one organ replaced by

another organ simply because you changed

the master regulator there so it gives

you versatility like for example I can

have this line and I can cross it with

another line where the UAS is for

another gene it gives you that

flexibility so I can have a library you

know like driver is this and this is the

UAS so like that I can have and then by

bringing them together I can activate

that so that's the main reasons the

previous one also it is the same so that

is because you you want to know in which

imaginal disc okay if I activate pack

six I will see this phenotype or rather

if I express pack six in different

imaginal discs what happens so that's

what you are testing you didn't get it

so see you want to know like two things

one where two things here one is what

does this protein do if it is put in a

particular biological context another

honest which imaginal disc has the

ability to develop what kind of

structures okay so normally it may

develop let us take this for argument

ant and our normally but it has the

ability to make ie as well if you

provide an eye specific transcription

factor so that is one thing

second this also tells which imaginal

disc can develop what structures so both

it tells about the master regulated like

what this tells you is a necessary and

sufficient condition for passive pack

six to develop i right and the second is

which imaginal disc is responsible for

what structure so both you can get out

of this so this has been extensively

exploited by the Drosophila

developmental biologists you will find

lot of papers you will hardly find a

paper where they don't use this method

alright so that ends this but we have in

free

so we have another lecture we can begin

which is basically continuation of the

same yeah see we already saw this yes so

this kind of tastes you this will be the

last concept for today we are going back

to the same pact sixth promoter region

okay so you need to remember this I am

talking about park six promoter so where

Pak six expression how it is regulated

is the focus so we are looking at its

own in answer I an intern PAC six when

it's produced it itself is a

transcription factor as well so that we

will see in the next slide so I don't

want you to get confused we are looking

at the promoter as well as protein of

the same gene so now we are revisiting

these different enhancers remember I

told you each one seems to be a

particular tissue specific expression so

that is being tested here in this

particular image where you are only

taking you know a part of it like you

are taking this these two enhancers and

the rest are not there but you have lag

Z reporter here and as a result you see

in the pancreatic region as well as

where the ie is developing lens and

cornea only there you see and you do not

see in the you if it's neural tube you

should have seen in this part so you

don't see it so this again demonstrates

the modularity of the enhancers so in

this particular gene it comes in four

modules okay when all four modules are

there it will be expressed in four

different tissues

spatial regulation is conferred okay so

this is modularity now we are going to

look at another concept of enhancer

function which is combination okay

combinatorial function so that is there

a little bit in this sequence where what

you're finding is to one particular

module a combination of transcription

factors bind and only when these two

transcription factors are binding to

this pancreatic module

build the pack six be expressed in

pancreas ok transcription factors act

combinatorially or in combination and

enhancers act modular e in a modular

fashion each model responsible or one

specific tissue so the odd is further

explained in this particular cartoon so

we are looking at two different genes

okay in one a target gene of pack six so

here we have got in the back six protein

and then we are seeing what it does to

the promoter of another gene it's a

transcription factor so to make this

particular crystalline expressed in the

lanes pack six has to bind it it's a

binding sequence and that is not enough

it has to have a combination of sox2 as

well as el mouth only when all three are

present this crystalline will be

expressed in the lens this combination

of transcription factors are required

okay that's why we say transcription

where does work in combination and these

other proteins shown here aid in these

interaction among these three

transcription factors when bound to this

promoter region and additional example

is here so matter of studying you know

the growth hormone it's produced in

pancreas where pack six binds to its own

binding sequence but then that promoter

should have you know other transcription

factors for example pd x and p bx

binding to their own respective enhancer

regions only when all three are there

this summit of strata in transcription

will happen so enhancers have modulus

and transcription factors work in

combination in each of those modules

this is clear so i will stop here or

yeah so this is summary of what I just

said

so combinatorial functioning of
transcription factors make coordinated

gene expression okay

so therefore like for example you may

have

under a particular condition you want to

activate certain set of genes in certain

parts and all of them can be handled if

you if that condition activates only one

transcription factor so that will go to

the right places and if the right things

are already there it will Express so

that coordinated expression is possible

okay it's a very sophisticated

coordinated expression compared to the

yeast gal4 or bacterial operon systems

okay where you have all the coding

sequence under one promoter but here you

have different controls and more

sophisticated coordinated expression so

I will stop here in the next class we

will get into transcription factors okay

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