lecture 3 9/24 slides two now

nuclear lamina and its role in chromain organization.

what infleunces trancirptional activity. hot topic, where scientist think that a gene depeonds on where in the nucleus is located. not all gene activity is controlled by cis elements. but its also spaitical postiion of the gene within the 3d nuclear space. in cancer and other diseases, those specific genes are hyperactivated . when u llook at the mtuaitono f the gene the scientst could not see any mutation but the gene will highly upregulated. like tumor supress gene was downreuglated even though there was no mutation in the gene. maybe its just not always the mtuation. but it could be where in the nuclus the gene is located.

nuclus of a human cell is 10micron in diamter. inside the 10 micron whre the gene is located is it pheripher or the inside that influences activity. …

some known factos that influcne trancirpioal actiity.

heterchormatin nuclear lamina and onctext of diseas and how in pogeria patients and how spacital postition of genes or chormain influcne activity. SOOOO heterochromatin /..

the nuclera peripheri is closer to the envelope, inside the nucelus there is dark regions these are high elecontron dense called heterochormin or condenseed chormatin. they are black colored in microscope. euchormtin are loose , deocmpacted, open chromatin.

general rule of chormatin compaction high condensed sems is trancirppally ianctive and open is trancirpally active. significant amount of trancioroally inactive chromin is located at the nucelar periphery.

blob inside the nucleus , there is a lighter shad area that is nucleuous there is an increase association of heterohcroamti or highly condensed chormatoin , that is becaus ethe nucleous is formed by a speficifc set of genes that are called ribosomal genes, genes which code for ribosomal rna.  they are located five different chormosome region in the human nucleus. the P arm of the five chormomes are enriched with ribosomal genes, ribsomes rn genes and those genes are highly enriched regions are also high heterochormin.  since neucleous are fomed by the organizaion of ribosmal genes the heterochormin which is part is allo reognaized aroudn it. consequence of how robsiomal rna geens to form a ncuelsu dictaties that structure .  

the nucleuous is the lighter less dense reigon inid ehte ncuelus. it is not srrouded by a mmbrna instease it formed aournd speicifr chfomromsal regions that contian RRNA genes. so inside the ncuelus are rRNA genes. those genes that code for rRNA are found on five chrmosmes. theyre located on the short parms of thsoe chormosmes. in regions called ncueloar orgnaization regions.

not true to the heterochromin nucleaur periphery  that are organized aroundthe envelope. 

how chormin is tructured and organized within the nucelus. so there is the outern ncuelar enveiole and inner, and the NPC structure going through. the outer nuceluar membrane which is  continoum with the ER. and risbomes attached to the ER.  the cytosplasm and the nucelopalsm, then theres the nucelar threa and basket. heterochormin is enriched ebenth the nucelar envelope , the work like structure are chormatin, heteorchormin. 

U chormin is decompacted. 

the nucelar lamina is beant the nuclear membrane, it isnt in yeast , present in higher eukaryotes. this proteinaceous structure located just beneath the nuclear inner membrane is why these heterochormin are condested at the periphery.  looking atht e nuealr laminar it is a mesh like structure made of proteins. inside the nucleus envelope. they are made of lamin proteins. there is a head domain that is n terminal c temrainl tail domain and the coiled coil rod domain. this structure of intermediate filemnt  proteins. like class 1,2,3,4,5 . lamin is a type 5 intermediate filament. all of the rest are locaies or distructed in the cytoplasm, only type of the intermeidate filament which is localzied insie is the laminate . the rest are in the cytoplasm. another intermediate common filament is called diamter of the structure is between mcirotubules and actine, there are three major cytoskeleton structure of our bodies, actin, microfilemtns,  microtubules. 

most well known is kertain that makes hair which is in the cytoplams. LAMIN is the ONLY in the NUCLEUS. since laims are inside it needs to have a NLS sequence, its a bigger protein and has a nuclear loczaiton singal. lamin proteins can itneract with eachother to make a homodimer type strucutre, type lamin genes that make this protein, there are a type lamine and b type lamine.    A type lamin , a single gene, can make two different isofomrs of the protien tha is called lamin a and lamin C. lamin a gene can make  mRNA, two differne tyopes of mRNA and can encode for A or C. … 

differential spliciing can do this

B type  lamin is made up of two different genes, lamin B1 and Lamin B2.  so three genes(lamina A and two Lamin B). different between lamins?

Lamin B which is made of 1 and 2 are expressed in every cell type in ur body. this is becaus ethose geens are active, transcriptally active, lamin A which makes A and C are expressed in highly differented cells or post mitotic cells the cells which stop diviidng.  they are the finished intial stages of division cyela dn post mytotic and differentedated.  

most cells in our body in an adult are highly differneiated cells , only a few cells have capbility to divie like stem cells. they continue to divide. those stem cells adult stem cells or embryonic stem cells are undifferneated cells. lamin a type gene doesn crepss in stem cells beucase they are undifferneiated. Lamin B gene is in both, differneiated, or undifferneaited. 

LAMIN A gGENE. the gene structure there are 12 wxons and the line between are intronic sequences, 5’ to 3’. LMAIn a mrna isoform has all twelve exons to make lamin a type   mRNA and makes a lamin A type protein and if it tranlstes to that , that exon gets tranlsted, some regions will be head domain, coil domain, tail domain , all thos part of them .  its a bigg protein 66a amin oacids. lamin c is produced from same gene  by differeintal splicing will contain exons 1-10. the last two are aprt of the lamin a type mRNA.  so they get spliced out of the tracnription during a post trancipraol event. its a smaller protein. Lamin a and lamin B have the caax motif(including lamin b1 and b2)

Lamin C lacks the caax motif because it is removed from exons 12. so since lamin c doesnt have the exon 12 it has no caax motif. 

Caax motif is helped fro anchoring onto the memrbane. protein can be anchored to the memrban strucutre, so lamin A and B proteins can be prenilated , have a fatty acid chain attached, and helps protein get anchored to the inner part of the ncuelar memrbane rather than th eplasm amemrbane. ..lamisn can be dimerized, dimer polymer and create a fibers. 

mutleiple lamins can itneract head to tail and head to head.  these fibers are about 10nm in diamter. 

Lamin a to b and to c. the fibers will form and stay close ot he inner nculsu membrane because A and B have the anchoring. 

if you generate anitbody against on the the  lamins like almin a and do a straining a confocal microscopic imge of the ncuelus, and see how the laim A protien is distrbuted.  u will see stainign. boundary of the ncuyelus, the periphery will be stained and see lamin A. 

Some differenence between the caax  between lamin A and B type. 

Some major mutation ahpepnign in that particular region in the lamin a gene that controsll this process. Lamin B 1 and 2 are the saem, there the  caax motif sitting in the e tmerianl of the protein the carboxy temrina, getrs prennihilated by forming at the end of the cysten  which is by far nuclei tranfer, and then it gets methylated, and then be anchored to the inner membrane. that s for lamin b2 and b1 classic lipid anchoring. But for almin A its different. Pre LAMIN a  … 

in lamin a prtein when its formed it has the caax mottif(not C) and it gest pharnyclated, and methylaed, bu afteer its donw, the lamin A , somwerhe upstream of the parhmycatlion is happning, will be recongized by a protease enzyme (FACe protease) will recongtize the astretch of the amino acid and cleaves. the protein so now you have two lamins A.  most  of the lamin is at the five prime end of the protein. and we call this mtuer amin protein and there is a sterch of amino acid c terminal which has the farnesil group. based on the funcitoality the lamin a protein is the  one that doesnt have the farnesily attachment anymreo. so it goes through the preoces and gest cleaved to make a functiaol lamin a protein that doesnt havge the anchor. we dont know what the small stercth of amino acid does. the actuall funcatil lamin A , funcatioal lamin doesn have the farnesil attahcmetn. Lamin B and B2 does have it. how does  the lamin A protein still stay at just the interor part of the ncuelar lamina or part of the ncuelar lamino. because it doesnt have an attachmetn and it doesnt have a fanesyl attachment onot it. . the reson why the lamin a can stay is becayswe it can itneract with the lamin B and form the ifbers. 

Also trye for lamin c which has no caax motif, it stays at the interior periphery because it interacts with the lamin B. So mature lamin a doesnt have the caax motif anymore, mature lamin 1 and b2 have the caax motif. 

There is a subset of protein inside the cell which also keep the lamina at hte nuclear periphery. this is called lamin associated protein. Inner nuclear memrban protein INMS. INMPS’s they are the same. These proteins are tranmemrbain proteins that are attached ot ht einner nuclear membrane that can itneract with lamins. Next elvel of attachment. it has other functions too . there are foru example. 

EMRIN, LAP1 whch is almin associated protein

LAp2beta and LBR , lamin b recepto. there are four of them example of inner nucelar membra protiens that intearct with diffene types of lamine. LBris a multi pass tranmemrbeain protein. dont remember that these are different types of INMP. they help keep lamina structurelaly formed. these structure can intearct with heteorchromin structures. so these protein the INMP’s as well as the lamins have high affinity to the type of chormin which are compacted now how do they do that because the way the y interact specifailly with hih compact chormisn is because the proteins like HP1 or BAF .specifially intearct with compacted chromain .   

hp1 and baf proteins binds to  compacted protein and has affinity for the INMP’s which they interact with the lamisl  

So these INMP’s are tkeeping the lamin at hte nucler periphery and those structures intearct with protein HP1 and BAf whihc have higher affinity for ocmpacted choarmin.  

hetochromin structuers are tranciprioally inactive. the nuclear lamina plays a key role in mainitn that division between hetoerhcormin and euchormin. not every chormin at the nucelra peruphery is inactivyt, its jsut a general tendency. Some genes XYZ postion at the pirphery are tranciprially active but most are inactive. Lamina funcion playrs a rol inst rcuture in keeping the ncuelar shape . 

mutation in the lamin protein a b c, if u distruct the laini strucutre the nucelus will not form the round structure, the perfect ncuelr shape reqiuires the laminar strucutre, it give suspport to the ncuelar membrane. lamin structure can orgnzie chormin in a way heteorhcormin to be located at the periphery. sceitnest were studying mutation associated with diseases , major cause of set of diseases always aossicated iwth mutaiton of losso f fucntion fo LAMIN A gene mtuation. lamin a gene mtuation are prevelant in diseases in humans called laminopathies .  laminopathy is a disease that is caused by a connection with mutaiton associated with LMNA lamin A. not clear why? lamin b has no effect, and not much of C. all of these seems ot be mutation in lamin a gene. 

lamin b may not have the same phenotype is because lamin b is expressed in every  cell type and loss of ofucntion in lamin b willr esutl in embryonic lethatlit. because it is essential to the gene. so loss of funcitonof lamin b will result in the emrbyonic lethality.

the organisml will not surive and die in early emrbyonic stage and u will not see the manfiestation of diesases. wherease l amin A is expressed only the differnetiated mostly differentyed cells. so durig early emrbyonic developemnt there is no differentated cell. so lamin A is not expressed so the emrbyo can reach specifc stage and then the disaese manifestion will come bcause of that. thats where the lamin A expressed. why lamin c doesnt shoa sever phenotype? idk

progeria is the premature. agtin disease, in the case of progeria patients, a sif number of mutations on dif exons which chnages the amino acid consitutiono r compostition is associated with progeria. dont remember all those amino acids subs.

lamin a gene is reiddcled with mtuations and can cause dif levels of porgeria. looking at one mutation, this causes a non functional a protein. normal nucleus structure is where the lamin stianign is present but if u take the cell from a progeria patitns, a GPS patient. you can see the ocmplete lamin structure . lmain which is present in the GPS patient is nonfunctional because the main role of the lamina is to maintain the nucelus shape. so the GPS patient will have a weird shape. lamin which is present in the GPS patients are non funcitonal.

A particular mutations, exon 11 and 12 is part of hte lamin A. in normal indivual 11 and 12 will splice togther to form a funcitonal mRNA. all these introns will be spliced out to proply form a functional limin A mrna. in the subset of the progeria patients, there is a mutation within a single nuceltodie in the exon 11 sequence, the single nucelotdie mtuation, there is a change in about 30-40% premRNA it gets abbrently spliced so the mtuaiton activates a cryptic splice site, new splice site. this region gets spliced with the atual splice site of the exon 12. so part of the exon 11 will be seen as an intron and be spliced out. this happens to 30# of patents .

rremember that there is c ryptic pslice cute and one nucletoide change and results in a new splicitng activated. and this exon level hass a loss of specific region.

lamin A gers cleaved alter, pre lamin. wild type lamin a, the prelamin a and gets cleaved and forms. afunctional lamin protien, but in the. pateints of progeria, the lamin A mutant, delta 50 lamin A protein because it lost some of the exon 11 and we called thos protein are called PROGERIN,. progerin is a mutant lamin A protien in these patients which lost htat amino acid sterch which part of th elast region of the exon 11. because of that the region is lost in patients has that phase protase cleave site.

the cleavage , phase protease cleavage site is lost in the progeria patients. with the mutated lamin A protien progerin. so there is no making of the functional lamin A .

the mutant lamin A protein, continue to stay on the inner nuclear memrbane because of the farincly attachment compared to the wild type lamin A.

this means lamin A protien, funcgtional lamin A protien can come of the lamin and come into the ncuelar plasm and play other funcitons. , it is associated to activate gene expression , dna replicaiton. but in progeria patients the lamin A protein the PROGERIN protein, is attached to the inner membrane , is a gain of funciton mutation, those proteins inhibit the lamin, it impacts the nucelar structure, the chromatin structure , and other types of normal funciton.

by pheontype they have abonral nuclear funciton, the hetoerhcormic strucutre is no more organized in the nucelar periphery. that means they are not able to organize it, many of the heterochormin structure tend to move into the interior part of the nucleus and reacttive trancirptioanly. scientist thing that progeria is a complex disease, intated by mtuations, it cold impacgt the nucelar strucutre, the chormin organizaiton, and make inactive hetoerchromatin to active, and make it trancitoally active.

dont know molcuelar mechansim of progeria protiens. for sure mutation of lamin A causes diseases, and syndroms.