Lecture 4 - 9/26

we talked about lamin and how nuclear lamin maintsin the nuclear shape, and chormaitn organization, by organizing heteorchormatin, compact ath the periphery, and saw progeria where lamin a is mutated and those lamin a become non functional. the pheontypes are werid. it also has impact on the cellular physiology.

several of the INMS proteins , LAPS, EMERIN, LBRS those inner nuclear membrane proteins interact with lamins and inflcue the heterochromatin structure, mutations in those INMPS show symptoms to laminopathis indiacting there seesm to be some connections. between lamins as well as INMS and their functional, and impacting mtuation in these genes resuls in similar phenotyeps.

how chormain organization impacts gethene expression. euchormtin is light color and in the interior part of the nucleos. basic unit of chormin is DNA. and proteins tha make the chormin. the major protein compeonet of the chromsin is histone. histone are basic protiens and small basic postively charged and part of the nucelososme structure.

the naked DNA is compacted at differnet levels or organized at different levels. different . the first elvel of copation is forming a ncueltosome.

naked dnais what ia forming a nucelosoems. this particular structure where dna is wraped around histone proteins to form what i called nucelsoomes . so thats the basic unit of chormatin. dna wrappiung around histone proteins to form nucelsomes. this is 10nm diameter. beads on a string structure, whihc is the histone proteins and the dna wrapped around. this is first level of compation. the next is 30nm compaction is wehre the nucleosomes and dna further compact at different levels to form a next high level of structure. further compaction happens but donw know how chromain that is going from naked dna goes to 10, to 30 , and goes further, just know it happens. why do we need dif levels of ocmpaction is because the average size of a nucleus is 10nm dimater. it has several meters of dna, if u take dna of every chromsome 23 pairs, and then tie them together its about 2 meters. DNA has to kept inside the 10nm diamter nucleus, so the dna has to be compact. the best way that cell can do this is by forming the intial nm and then 10 to 30. in every cell in our body many of these genes needs to be tracnriptionalluy activted, for a gene when it becomes tranciprioally activate is has to remoe the nucleus from the gene to keep the dna naked to let proteins like polymerase to tracnribe. the dna needs to be stripped

it has to happen at a faster pace. heat shock re[ponse, there is a set of heat shock geens that get activated and the product is chaperones and their job is for the cell to not go into toxic shock . they facilitaty processes to make the cell survive, help. this happens to happen in miniutes, so if the genes are inactive they need to change the chormaitn structure and open up the chormtin of the specific gene and this needs to be fast. even tho these things are ocmpacted it needs to be acessibel

whats the basic unit of a chormtin, it is called nucleosome and they are dna wrapped aroudn hsitone protiens. chormain means dna and proteins. major component of hte chromain is histones, small part of proteins are non histone proteins. many of the TF or even polymerase which are bound ot the chormin infleunce chormins tructuer and are chormin associated proteins

so nucleosomes is a dna wrapped around histone protiens. it is highly evolutionarly conseved strucutre, the size of the dna is about 147 base pairs. there are about eight histone protiens. eight molcules of histones are preosen in that core hsitone structure. four types of hisones . H2A. H2B, H3, and H4. this makes up nucleosomes. the dna sitting ouside of the core hsitone, the cor enucleosome sturcture is called linker dna because that part of the dna links the nect part of nuceoom,es. so two nuceoomes sitting next to eachother and the dna connecting to these two are called linker NDA> the DNA size can vary between organized, this isnt fixed.

Ths hsitone that interacts with the linker dna is the H1. its called linker histone too,interacts with the linker dna. it isnt part of the core nucleoeomes. its outside the cor enucleosome. this structure is part in every chormin regardless if its euchormatin or heterchromine.

scinetist though that these nucleosomes is to compact the DNA so it can accomdate the DNA, boring. just to keep it inside the nucleus, is boring. when trancirption happens there are change that happen. there are post tranclation modifications of hte proteins that are part of the nucleosomes, histones. major changes on specific regions called tails. Histone tails. the n terminal region of these proteins , amino termal is about 30 amino acids in size where are unstrcutured . histones sit inside the histone and hte n terminal region flex out,tend to come out of the DNA. in the case of histone 2a and 2b they have. c terimal taiol and the n ternal but h3 and h4 have only the n terminal. histone one also has a small tail. the sequences of the tails can get modified postr translationally, phorylation. or acetylated, methylated, ubiqualated. dont remmeber all modifications

all modification have important funciton other than compaction. modification can influcen compaction rate, hisone four tail can be aceytaled , specific amino acids, LYSINE that has a basic postiive charge. it has a postive charge and interact with dna more stronly at part of the nucelosome, and interact with negative patches with neighboring negative patches of nucleosomes

postive charges of hte lysin interact with engative patches in neighboring nucleosomes. like H2H2B whihc have. negative charges and dna has negative charges that will intearct with the lysin so form a negative postive chargvge interaction. this helps reduce the distance between two of the nucleosomes. when ever nucleosome does this is decreases the distance bewteen two neighboring neucloeomes. thats fist level of compaction. internucleosome interaction reduces the distance. when those histones h4 get aceylatied, it neutralizes the charge and the tails now reduces the interaction and reduces the nucoeome distance. aceylation opens the chormatin more, increase the decompaction rate. so many lysines in histone 4,3,2 and they can get aceytlated, in general the lysin on the nt erminal tail which have postiive. charge tend to facilitate chormatin compaction and aceylelation will open the chormtin up and increase the decompaction.

at a single lysine level its a small efect. similar thing happen in different modification which are present on same lysine histone or same nucleosome can then impact can be major.

changin the charge or neutralizing the charge isnt the only wya. modifing the tail regions can also act as bdingin sites for a group of proteins. like phorylationo f a protein can change the conromation of the protein and now the phroyatled sites act as bidning sites. like RTK receptor is phroylated and now acts are a region of binding site for hte GRB2 protiens. simialrly the n terminal. regions which have different modification post tranlational can facilitate or inhibit depends on the contact binding of a specific target protiens. these proteins could change the chromatin strucutre. so based on the modiciation and impact on gene expression, there are specific modications that have a psotive or negative effect on comapctions or decompaction. only modications to remember is the histone 3 modification whihc is called hsitone 3 lysin 9, the 9th amino acid of the histone 3 is a lysine. this lysin 9 can be aceytalted or methylated. either or not both at the same time. this seems to eb a charctist region of chormatin which are highly compacted or hetoermchoamtin. when aeytalted it beocmes part of the open chormtin. methylation will make it more ocmpact. histone 3 lies 27 and again aceytaltion is open and methylation is more closed. them there is histone 3 lysine 4 and mehtylation is part of the euchromatin (the opposidte and more open). when methylation of a lysin it is context depend and which lysin gets methaiteld 9 27 or 3

methylation is context dependent on what lysin is methylated. no rule on methylation but aceylation does have a generic rule. acetyalted is euchormatin and deactylaed are chomatine.

lyein four mehtylation is part of the open chormatin unlike 27 and 9. when a particular lysin it depends on which lysin will have an impact. acetyaltion is trict rule. there are enzymes that modify these amino acids, the enzymes that add methyl group are histone methyltransferases. then ther eis histone acetyltranferases. those enzyme that remove methyls are demethylationa dn then there is deactylations.

those methyltranferases that add to the lysin h3 are differen from the transferase that add h3k4 methylation. so differen methylasses for different tails. There is HAT histon acetyla tranferse which add acetyl group to specfic histone tials. some of histone aceyltranferases can acetyalte non histone protiens. KAT and HAT is the same thing. HMTA hsitone methyl transferases. example how these methylations can infleunce chormatin structure …

open chormtin, 6 nucoelones and in reponse to a specific singal the cell wants to inhibit trancirption of a gene, that means it has to change the chromatin structure.

the cell will recruit specific methyl tranferases like H3K9 and add a methyl. H3k9 methylated , u can methylate the same lysin and add two three methyl groups and its called monomethylation, dye, and tri. most of the repressive funciton is assocated with the tri.

higher chromtin compaction is achieved by trimethylation. h3lysin 9 tri methylated, the methylation doesnt change, the methylated sites will be recongized by a group of protien called HP1 and it will recongize the H3k9 trimethylated nucleosome regions.HP1 has the chormo domain and chromoshadow domain. The chromo is what binds to the trimethylated nucleosome and bidns to that region, the chromo shadow will interact with other proteins that have chromoshadow domain. so if you have two hp1’s next to eachother their chromoshadow doamins will interact with eachother. so hp1 can recruit more hp1 because of the chromoshadow domain.

hp1 proteins sitting iwth eachother will interact and act like a glue and prevent the interaction of other proteins into the regions. hp1 hp1 interaction will increase the chormain compaction and make it more condensed.

HP1 was used in a dif context in lamin. we said that lamin bind to heterochormin through its interaction with HP1 protein.

in the nuclear membran protein it can bidn to the hetorhcormic region because it can interact with HP1 which is part of the heterhcormatic structure.

heterochromatic structure, the HP1 protein which will bind to these H3 lysine 9 methylated regionwhich is characterist of the compact chromtic will make the Hp1 bind and make the chromin becoem condensed and it the bidning of HP1 will make it ineteract with the perihperhy and interact with lamina, and lamin associated proteins. so Hp1 is a characterist of highly ocndensed chromin.

regions that are nucleosm efree or contain other non nucelosomal proteins so those canno tbe methylated by the h3k9 methyl transferase, because they dont have histones so it cannot spread. every chormosm is made of singl dna and 23 pairs, and every chormosn is linear dna. so we need thigns to prevent full heterochorminzation.

this compact chormoin structure will prevent the interaction of other molcules in that region including trancription factors or other things . is make chromin caompact.

another fe3ature of heterochromainzation. it can spread to both sides of the chormin, you can spread that to either side of the chormin. hp1 will recruit more methyl transferases like h3k9 contain chormoshaodw domain,a nd if a protien has. chormoshadow domain, if hp1 can bind to another hp1 it can also bidn to tnaother protein which has a chormo shadow domain. so since h3k9 emthyulatranferrase ahs the chormsoshaodw doamin it can recruit that region .

you take aceylation as an exmple of a nuceolsomes, histone 3 or 4 , it is catlayzed by HATS or CATs and decyatlion is done by deacetylation enzymes. in genral HATS will condense and HDACS will open.

eueurchormin and look at the histone tails have acetyl modifications. if u look at the heterochromin have less aceytlation. and more methylation groups like h3lsin9, 27, h3k20, and there ar emore.

modification has a direct role . bromodomain looks at acetylation reciton. if a protien has a bromcoodmain and thos eprotins have higher affinity to histone tails that are aceytlation. many of the bromodomainds are part oaf the open chormin or eukachormin wehreas the chormodoamisn is part of the heterochormin or condesed chromin

So if it brings there, those methyl

transferases will add methyl trimethylated sites to the

neighboring nucleosomes and that now recruit HP1

and it does the cycle will continue till a point where

you have regions which are nucleosome free what

we call boundary elements that mean boundary elements

are regions which are either nucleosome free or

contains other non-nucleosomal proteins so those ones

cannot be methylated by H3K9 methyl transferases

because it's they don't they are not histones so then

the spreading will not happen. Because otherwise,

the complete chromosome will get

spread into heterochromatin. Remember,

every chromosome is made up of a

single linear DNA. Remember that.

Right? So if it's 23 pairs of chromosomes in our

body, it means every chromosome is nothing but a straight,

a linear DNA where nucleosomes are attached to.

So you need to have something to prevent

those heterochromatinization. Otherwise,

your complete chromosome will get heterochromatinized

as part of the same by using the same principle.

Okay?

Any questions?

Yeah,

sorry.

I think you just use exactly

opposite of what I just told.

Acetylation, it basically destroys the

charge, which will increase the distance.

Or which will aid the distance. Right.

The charge, positive charge, will facilitate

more interaction with DNA as well as what is called

H2H2B negative patch, which is negatively

charged path, which will more interaction of the

charge helps them to increase the compaction.

So, neutralization of the acetyl group, the

neutralization of the positive charge basically increases

the distance, which decreases the compaction.

Any other questions?

Okay. So, I'll go a little more into,

I mean, it's basically a continuation of the

same topic. Again, as I told this earlier,

that when you take acetylation as an example,

acetylation of a nucleosome, in this case, whether

it is histone 3 or histone 4, it does not matter.

It is catalyzed by histone acetyltransferases

enzyme, HATS or CAT, it does not matter.

Whereas, this deacetylation is done by histone

deacetylase enzyme. So, there are histone acetyltransferases,

different types. histone deacetylases that

are a different type, they can change. By controlling

the acetylation, you can control the chromatin

status, and these enzymes play a key role in that.

And again, so in general, histone

acetyltransferase will open up the chromatin,

H-tax will increase the chromatin compaction

or decreases the, what you call hypoacetylated

histones tend to be a characteristic

feature of more compacted chromatin, right?

Now, as I told, because of different marks like when

you think about acetylation we know. I think it's more

clear you know if you have a U-chromatin they tend

to have more acetylated nucleosomes whereas if it's a

heterochromatin you will have more

deacetylated chromatin but methylation

depends on the specific methyl sites.

So what scientists know is if you look at a U-chromatin

region, if you take a U -chromatin region, open chromatin

region and if you look at the histone tails and

look at the modification, they tend to be enriched with

acetyl modifications, right? And some methylation,

because, for example, H3 -lysin-4 modification could be

characteristic of euchromatin. But if you look at the

heterochromatic regions, they tend to have less amount of

acetylation in general. That's why we call hypoacetylated

regions. Then also they have more methylation because

there are more methyl groups, more H3-lysin-9, H3

-lysin-27, H4-K20, there are many more. Those are methylated

as part of the heterochromatric region now it's

the question is is a cause of consequence this modification

how does it help it does have a direct role it's

not a consequence of heterochromatinization because as

i told by using h3k9 trimethylation as an example we

saw that that that modification helps to increase the

chromatin compaction by recruiting hp1 protein so similarly

H3-lysin-27 or H4K20 methylation, those marks also

recruit proteins like HP1, not HP1, but other proteins

and that also helps chromatin compaction or

heterochromatinization. So it's not a consequence, this modification.

These modifications have a direct role in changing

the chromatin structure or changing gene expression

by recruiting specific factors to that region, right?

So that's what exactly is shown here.

That, you know, for example chromodomain of a

specific target protein can bind to this modified

region and then can change the chromatin structure.

Bromodomain is another word, just like chromodomain,

bromodomain is specifically looks at acetylated

regions. So if a protein has a bromodomain, then

what that means is those proteins may have a higher

affinity to histone tails which are acetylated,

which are more open chromatin. So as a result, many of

the bromodomain containing proteins are part of

the open chromatin or euchromatin, whereas most of

the chromodomain containing protein seems to be part

of heterochromatin or condensed chromatin. Okay.

Okay.

I will stop here and then in the next class

we talk about how these modifications can

change the chromatin structure and how that

influences transcriptional activity. That's