Dosage compensation

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  • Created by: Sarah
  • Created on: 17-05-19 18:23
what are the 3 strategies for dosage compensation?
1) Make X chromosome in M twice more active 2) make one X chro in Fs (hermaphrodies) totally inactive 3) make both X chrs in F (hermaphrodites) twice less active
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what animals double the X chromosome activity in Ms?
drosophila
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what one makes both X chromosomes twice less active?
C elegans
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what makes one F X chromosome totally inactive?
Mammals
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what chromosomes are presrnt in the same number in Ms and Fs?
autosomes
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what is dosage compensation?
the mechanism to equalise gene expression from X chrs in Ms and Fs
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what is lethal chromosome numbers?
any wrong amount of chromosomes eg monosomies and trisomies
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in drosophila how do they get equal X chromosome expression?
make the male X twice more active
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how do make the M one twice more active?
express dosage compensation factors in a sex specific manner
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who expresses sex lethal?
Females express sex lethal
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when is sex lethal activated?
early in development in animlas with 2 x chromosomes
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what is sex lethal?
a splicing factor
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what establishment promoter does sex lethal expression come from?
SxlPe
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what happens when sex lethal expression from sex lethal maintenance promoter is activated?
the sex lethal protein promotes splicing so that translation termination exon 3 is excluded and taken out
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what does exon 3 being kept in and not spliced out in M drosophila mean?
translation is terminated and functional sex lethal is not made
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what does the functional sex lethal (only in Fs) do?
maintains its own expression and prevents translation of MSL2 (male-specific lethal-2) protein
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how does sex lethal prevent translation of MSL2?
by binding its 5' UTR and 3' UTR
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in Fs is the MSL complex on or off?
off because its male specific
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what is the series of events that leads to MSL complex being on in males?
no sex lethal to splice out terminating exon 3 -> no functional sex lethal -> MSL2 is kept on -> MSL complex on
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series of events that leads to Fs MSL complex being off?
sex lethal made bcos have 2 X chrs from sex lethal establishment promoter-> sex lethal splices translation terminating exon 3 -> function sex lethal -> sex lethal prevents MSL2 translation by UTR binding -> MSL complex off
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simple organisms like nematodes have how many chromosomes?
X0
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why is it a problem that Fs have 2 X chromosomes and Ms only have one?
imbalance in copy numbers so double copy of the genes on the X chromosome while males only have a single copy of the gene (1 X chr)
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what is downs syndrome an example of?
trisomie of chromsoome 21- chr is small and number of genes duplicated not that much so not embryonic lethal, developental probelm when too much/little of product
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what dosage compensation mechanism is used is nematodes like the C elegan?
both X chromosomes in females made twice less active so half activity of both
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what are the 1 of 2 promoters that sex lethal is specifically expressed from?
establishment promoter or maintenance promoter
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what promoter is used specifically in Fs? what does this result in?
sex lethal establishment promoter- result in the production of sex lethal only in Fs activated during development
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how does sex lethal maintain its own development?
later in develeopment the expression of maintenance promoter is induced
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what happens if no sex lethal is made?
3rd exons not spliced out- so there's premature truncation of the protein
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if there's functional protein from the establishment promoter what happnes?
its a splicing factor so it regulates splicing binds to Poly u stretches in its own RNA and induces a splicing change to exclude the 3rd promoter so functional sex lethal is produced
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what 2 things does sex lethal regulate the expression of?
1) its own expression- makes more functional sex lethal 2) MSL2- male specific lethal
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how does functional sex lethal regulate the expression of MSL-2
binds to its ' and 3' UTRs so it can't be translated
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what is the MSL2 complex?
activates male X's twice as much- a group of proteins which all bind together and it's essential for dosage compensation and viability and is has 3 mian components
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what are the 3 main MSL protein components?
MSL1, 2 + 3
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What does MSL1 do?
scaffold protein which brings components together
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What does MSL2 do?
essential for assembly of the compelx but is also a ubiquitin ligase
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what is MSL3 for?
binds trimethylated lysine 36 on histone 3 H3K36- histone mark of active genes
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what is MLE?
RNA DNA helicase
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what is MOF?
Acetylates histone 4 on lysine 16
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what is another component of this complex?
Rox
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what does rox do?
are 2 different long non coding RNAs- have disimilar sizes and sequences but they;re both required for bringing the complex together
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what is this MSL2 complex present on?
bodies of active genes on the X chromosome- this complex is only present and assembled in Ms so its present on bodies of active X genes in drosophila Ms
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how is this complex recruited specifically to the X chromosome and how is it spread to be localised throughout the chromosome?
MSL3
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what system was used to understand how MSL complex is recruited to the X chromosome?
drosophila polytene chromosomes
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what did the drosophila polytene chromosomes lead to?
led to a lot of discoveries on understanding how chromatin functions and is organised as these chrs found in salivary gland 3rd star instar dros larva really active produce lots of pro
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what is unique about polytene chromosomes?
there's chromosome replication without cell division as it enables lots of proteins to be made, when replicated stick to each other indiv chromatids condensed close to each other- really big bcos of this
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what can easily be seen on polytene chromosomes?
condensed and non condensed chromatin- lighter and darker bands
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are polytene chromosomes easy to prepare?
yes take salivary glands from drosophila instar larva squish them and stain using antiboides where diff proteins are localised eg associated with inactive chromatin
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where does MLS3 protein antibody localise to?
it decorates specifically the X chromosome in males
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how did they discover that MLS3 was involved in recruiting the MSL2 complex to the X chromosome?
was localised there with antibodies & MSL3 mutants the complex forms but fails to spread but is localised to about 150 distinct sides to the X chr- found in very specific band = no spreading
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how did they know that the MRE was the DNA binding motif?
they put it on the other chromosome and it could also recruit MSL complex which suggests this motif is sufficient to recruit MSL complex to DNA
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what did they use to find what the complex is binding to?
chromatin immunoprecipitation- MSL2 chromatin immunoprecipitation- MRE motif was enriched in precipitated DNA = suggests it might be the binding motif
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what is the MRE stand for?
MSL recognition element
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where is the MRE enriched?
on X chromosome in drosophila = explains why the complex is recruited predominantly to the X chromosome
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what does MRE contain?
known recruitment sites for rox1 + rox2 (long non coding RNAs)
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how does MSL3 cause spreading along the chromosome?
MSL3 caps spreading by binding to trimethylated lysine 36 on histone 3 (H336) = on bodies of active genes
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what else helps the spreading of the MSL complex?
ROX3 non coding RNA- mechanism currently unknown
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what does MSL complex need to do as its spread along the X chromosome
needs to increase the gene activity of the X chromosome genes by 2 fold so that males have the same expression as females
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what does MOF do?
acetylates H4K16
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how is gene activity increased?
1) MOF acetylates H4K16 2) MSL complex recruits topisomerase 3) MSL2 ubiquitinates H2B (only in vitro)
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what does it do by MOF acetylating H4K16?
weakens the repressive internucelosomal structure so opens up the chromatin and makes DNA more accesible for trancription
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what does recruiting topisomerase 2 do?
relaxes the torsional stress of the chromatin on the X linked genes, making chromatin more accesible and active
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what does ubiquitinating H2B do?
only shown in vitro- faciliates methylation of lysine 4_ 79 on histone H3- thought to be important for effective transcritpion elongation
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so what is the overall activity of MSL2 complex?
to increase transcription of X chr genes in M so on active gene bodies it modifies chromatin structure makes it more accesible to facilitate elongation by imporving speed+processiviyt of RNA pol 2
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what happens in mammals?
a single X gets inactivated
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what is the inactivated X chromosome in Fs called?
the barr body
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what is the X inactivation process in mammals called?
lysonisation
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is X chromosome inactivation in mammals a random event?
yes- occurs in epiblast afgter one X chr is inactivated its inherited by all progeny cells
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why do you see patches of orange and black in only female cats?
as X linked colour determinant gene which X chromosome was inactivated produces clones of red or black colour cells
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was is the master switch locus for inactivating the X chromosome?
The X inactivating centre (Xic)
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what is the X inactivating centre?
a single cis-acting master switch which is essential for silencing the X chr in cis which meaqns it silences same chr where its active and ensures initation of random inactivation
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3 key genes?
Xist ,Tsix, non coding RNA
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what does Xist encode?
non coding RNA
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what gene is complementary to Xist?
Tsix (xist name reversed)
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what does expression of Tsix or Xist do?
expresion of one inactivates and switches off expression of the other
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what is Rnf12?
protein coding gene = encodes ubiquitin ligase- an Xist activator
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what does Rnf12 do?
ubiquitnates and leads to degradation of Rex1 protein
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where is Rex1 found?
at the promoter region of these 2 non coding RNA genes
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what does Rex1 do?
simultaneously activates Xist and inactives Tsix
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how does X chromosome inactivation get started?
autosome provides a signal that promotes Tsix expresion whereas X chromosome has Xist and Tsix genes whcih are exclusively active
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what does X linked locci promote the expression of?
Xist expression
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the autosomal signal represses what?
represses Tsix and at the same time Xist linked locci promotes Xist and stocchastically probability that one chromosome when lot fo prodcution of X linked factor
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why is there a probability that on one chr there will be a lot of production of the X linked factor?
because both X chromosomes are active at the beginning so there's a lot of RNF12 produced so it binds to Xist prmoter and activates Xist expression which will inactivate single X chr
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why will the amount of produced X linked product RNF12 then get smaller?
now only one X chromosome is active so relative amount of Tsix activated product from the autosome will be higher than the X linked product which reinforces Tsix expression on the remaining active X chr
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what are both autosomes and X chrs doing in the beginning?
2 autosomes + 2 X chromosomes active- produces proteins autosomes activating Tsix and X chrs activating Xist. These proteins compete agaisnt each other random chance one X lot of Xist prmoting product will inactivate 1 X chr
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what happens when the X product is only from the 1 remaining active X chromosome?
concentration becomes smaller and the Tsix promoting product from autosomes is not affected still from 2 chrs so now theres more product than x chr one which ensures Tsix on remaining active Chr
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why cant the remaining active X be inactivated?
theres very little RNF12 left and a lot of protein makes sure the X is still active
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What is Rnf12?
ubiquitin ligase- the first known Xist activator- ubiquitinates and degrades Rex1- activates Xist and inactives Tsix
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when they looked at mouse embryos what did they find?
mouse embryos at early stages they found in most cells in F embryos would have 1 active and 1 inacitve X chr but some cases where both X chrs remain active or both X chrs become inactivated
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what does this mean for the genes?
in these chrs there's the wrong dose of x chr localised genes which means they have the wrong amount of products of these genes and over time cells die or outcompeted by cells where correct inactivation of one of the X chr so only these cells remain
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how does it propagae throughout the X chr?
Xist encoes non coding RNA which is associated specifically with inactivated chr so its pretty much the only gene expressed from the inactive X chr
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what does Xist non coding RNA do?
doesn't directly contact the DNA but it's needed for the association with C chr mediating proteins such as hnRNPU and YY1
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What happens when they translocate the Xic locus to the autosome?
it can recruit all the machinery required for dosage compensation but it cannot spread so it can recruit Xist RNA but cannot spread from locus or induce inactivation of whole chr
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what does this mean?
there needs to be booster elements enriched on X chrs specifically to allow spreading from this initial locus
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we don't know what the booster elements are but what might it be?
L1 long interspersed repeats- LINE-1 repeats
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what are LINE-1 repeats?
very common retrotransposon like elements in human and mammalian genome
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where are LINE-1 repeats enriched?
in chromosomes
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what is the indirect evidence that LINE-1 elements are involved in spreading?
there are bursts of LINE-1 expression from inactivated chr at the time of inactivation and they might produce local short RNas that facilitate spreading (hypothesis)
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what is the inactivated C chr organised in a similar way to?
constitutive heterochromatin- depleted of active chromatin marks such as axetylation of H3K4 me23 methylation and enrichment of inactive chromatin marks
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what inactive chromatin marks is it enriched in?
H3K27 trimethylation and DNA methylation and its known to recruit the polycomb complex.
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what is PRC2 involved in?
the repression so inactivation of expression silences
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where is PRC2 recruited to?
inactivated X chromosome which is Xist dependent thoguh it needs Xist but there seems to be no direct binding- mechanims unknown
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what are the different proteins such as PRC2, Xist RNA and different chromatin marks the same as?
same as in interphase and during cell division in metaphase to contribute to the inheritance of inactivation status to ensure that the same X chr and only one X chr is inactivated following cell dicision
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does all the X inactivation processes happen at the same time?
no in mammals doesn't occur at once. In mice for example some Xist RNA domains establishment or RNA pol depletion from X chr happens early some events later eg actual DNA methylation of CpG islands on X chr so its not all at once continous process
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how do C elegans reduce activity?
reduce activity of both X chrs
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what is an example of nematodes?
c elegans
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how is dosage compensation in nematodes similar and not to mammals?
dosage compensation is achieved through reduction of X chr is activity but there is no selection between 1 or other of the X chrs- both X chr activity is reduced
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how nematodes very similar to flies?
the process starts with sex speicfic expression of components of proteins
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in C elegans hows dosage compensation achieved?
by expression of particular molecules from X chromosomes and particular molecules from autosomes
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how many sex determination elements do C elegans have on the X chromosome?
4
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What are the 4 sex determination elements in C elegans called?
XSEs and 1 signalling element on the autosome
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what does the 1 signalling element on the autosome mean?
in males there'd be double expression of autosomal elements relative to X chromosome linked as there's only one X chromosome
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what would the dose ratio be like in Fs?
there would be a similar dose of expression from 2 autosomes and 2 x chrs in Fs
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What do X chromosomal and autosomal elements do to each other?
compete with each other by regulating the X-ol1 gene
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what do autosomal elements do to the Xol1 gene?
promote it
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what do X chromosomal elements do to the Xol1 gene?
inhibit it
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in C elegans with 2 Xs what are they?
not F, they are hermaphrodites X0- is male so they don't have male specific chr
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so in hermaphrodies what happens?
there's twice more products of X signal elements expressed from X chromosomes so one gene is repressed whereas in M less repressing leements from single X chr so 1 is active
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what is the protein that is a kinase and what does it inhibit?
XOL-1 is a kinase from single X chromosome is acitive it inhibits SDC-2 protein which means XOL-1 is only present in M
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where is SDC2 only present?
Fs
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what are the 4 genes on X signal elements that repress expression of XOL-1?
sex-1, fox-1, ceh-39, sex2
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what is on thr signal element?
Sea-1
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what does sex-1 do?
encodes a TF that activates XOL-1 expression
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in hermaprhodites (kinda Fs) how mcuh more products of X signal elements is expressed (that repress XOL-1)?
twice more
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in what sex is only XOL-1 acitve?
males
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what does the kinase XOL-1 inhibit?
SDC2
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why is SDC2 only present in Fs?
because in Ms XOL-1 is inhibiting it
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what complex is required for dosage compensation in C elegans?
DCC complex
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what is SDC2 found in hermaphrodies) needed for?
a regulator of DCC is absolutely required for the assembly of DCC
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where do other components of DCC come from?
maternally supplied
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what do they form
a complex similar to condesnin
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what is the condesin complex conserved in?
all eukaryotes
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what is condensin complex in DCC essential for?
proper chromosome compaction and segregation during mitosis and meiosis
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are the maternally supplied ones present in Ms and Fs?
yes
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what is the difference then?
only in the presence of SDC2 (Fs)- which enables hermaphrodies to assemble the complex
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why is the condensin complex essential for life?
without it chromosomes wouldn't be segregated and condensed during cell division
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because of the similarities with the condensin what is the DCC complex involved in?
partial condensation of X chrs and hats why their activity reduced
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what does condensin and DCC complex share?
share some of the same components all 4 of the DCC components except DPY-27 are components of both
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what did they find about particular X chromosomal regions that are required for recruitment and translocation of these regions?
they're sufficient for recruitment of DCC and further mapping using CHIP seq identified small reegions enriched in 12 bp DNA motifs
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what was the 12 bp DNA motifs identified using CHIP seq called?
rex sites
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what are rex sites necessary for?
are necessary for DCC recruitment on X chromosome
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how long apart are rex sites on the X chr?
about 100-300 sites distributed along the C elegans X chr
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what is different about the C elegan model?
there's no evidence of non-coding RNA being involved but again absence of evidence doesn't mean there are none
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what is not known about C elegans dosage compensation?
how DCC spreads from the intial recruitment site but accumulates along X chromosomes especially at promoters of actively transcibed genes- spreading doesnt seem to be dep on any propery of X linked DNA seq
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what does this all do to gene expression?
diff histone mods eg methylation of H4K20 is associated with gene repression and is enriched on X chrs in hermaphrodites so DCC complex reduces activity 2 fold
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what is not known about C elegans dosage compensation?
how DCC spreads from the intial recruitment site but accumulates along X chromosomes especially at promoters of actively transcibed genes- spreading doesnt seem to be dep on any propery of X linked DNA seq
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what does this all do to gene expression?
diff histone mods eg methylation of H4K20 is associated with gene repression and is enriched on X chrs in hermaphrodites so DCC complex reduces activity 2 fold
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what animals double the X chromosome activity in Ms?

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what one makes both X chromosomes twice less active?

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Card 4

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what makes one F X chromosome totally inactive?

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Card 5

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what chromosomes are presrnt in the same number in Ms and Fs?

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