X Chromosome Inactivation Flashcards
Describe X chromosome inactivation
All about non coding rnas
During embryonic dev, early on
In females, inherited epigenetically, stable throughout dev
Classical epigenetic pehenomena
What is the goal of x chrom inactivation
Deals with gene dosage competition in species with dimorphic sex chroms
- males and females have diff sex chromosomes
Dimorphic sex chromosomes lead to dosage imbalances between homogametic and heterogametic sexes
Organs have evolved different strategies to deal with this
Describe how drosophila compensate for gene dosage of dimorphic sex chromosomes
Male X gets hyperactivated = 2 fold more
Describe how mammals compensate for gene dosage of dimorphic sex chromosomes
Inactivation of one x in females, completely shut off
Name general properties of Inactive x
Physically compacted into giant chunk heterochromatin - visualize it = Barr body
Compact structure
Nuclear periphery - general property heterochromatin
Epigenetically inherited
Dna meth (promoters) high on inactive x
H3k27me, h3k9me, hypoacetylation of histones = all associated with heterochromatin - not much transcription
What is distinguishing feature of inactive x chrom
Xist rna = coated with non coding rna (xist) - fundamentally how inactivated and establishes properly of inactivated x
When does x chrom inactivation occur - gen
Randomly in embryo - coincident with onset of differentiation, early stage + embryonic dev
In blastocyst stage = when difffereniation, gastrulation
When does x chrom inactivation occur IN TE
First specifically in te - extra embryonic tissue
Specifically affects paternal x - imprinted
When does x chrom inactivation occur IN EMBRYo
After implantation, when epiblast cells begin to differentiation - gastrulation
How is x chrom chosen to be inactivated
Choice of which x is random = chosen randomly
Does x chrom re activation occur
In primordial germ cells
So x inactivation reversed, corresponds which migration to genital ride - global erasure dna meth, also leads to reactivation of inactivated x
Describe x chrom inactivation in cats
Randomness - can see phenotypically
X linked gene for co-colour = heterozygous on z chrom = patches of pigments of 2 diff colours
Recall - imprinting, issue of picking a chrom, embryo more complicated
What is mosaicism
Mosaicism is epigenetic NOT genetic
Name 2 key concepts in x chrom inactivation
Counting
Choice
Describe counting - x chrom inactivation
Cells can count, only one active x in female embryonic cells for everyday diploid complement of autosomes
1 active x for 44 autosomes in human, excess x’s are all inactivated
If engineer cells - excess of one active x inactivated- if create with >2 x’s = all x’s besides one inactivated
Describe choice - x chrom inactivation
One particular x chrom chosen for inactivation in each female embryonic cell, once choice is made - this x is stably maintained in the inactive state through cell divisions and development
What is XIC
X inactivation Center
Look at situations where defects/deletions on x chrom = effective in mapping region important for x chrom inactivation
- study x chrom truncations and translocations
What happens if x chrom some has no xic
x chroms missing x inactivtion Center cannot be inactivated
What is key part of xic
Xist = silencing caused by expression fo xist
Xist = single non coding rna transcript
What is xist
Long - 17kb, non coding rna, spliced (introns) and polyadenylated (3’ end) but retained in nucleus (no export to cytoplasm or transcription)
Rna processing steps after transcription =similar as other rnas
What does xist do
Coats inactivated x and causes silencing - turns into heterochromatin - IN CIS (means that silencing occurs on chrom it is expressed form, works on x that produces it)
Is xist found in autosomes
NOOAOOAOAOOAOOA
Move it exp = move xist to autosomes = inactivates it
What is a great way to study x chrom inactivation
Cultured es cells
Describe ES cells
Take cell out blastocyst embryos and culture in vitro
Derives from blastocyst stage embryos before germ layers are formed = pluripotent
Maintain properties from it
Cultured and differentiation in vitro = gives them things to stimulate differentiation in embryo
Describe exp - following x chroms, results -es cells
Undifferentiated = 2 active x
Differentiation = one x coated with xist
Differentiated= one active x, one inactive x
To do exp = hybridize probe to genes on x chrom
What regulates xist - generally
Involves interplay between cis factors (on x) and trans factors (autosomal)
What is xist regulated by - transcription
Antisense transcription
Gene that overlays xist in other direction = tsix - non coding rna
Downstream of xist, and then goes all the way through xist = transcribed in opposite direction
Describe how to visualize xist and tsix
Hybridize probe only to one strand or other - to part fo genome
Xist = detected by sense probes
Tsix = detected by antisense probes
Ex = when xist not there but tsix is = recognize some transcription but not xist
What is tsix
Relative regulator of xist
Normally x chrom inactivation in diffeerniating female es cells occurs randomly on either x
But when remove tsix promoter = changes
What happens when remove tsix promoter
Delete promoter of tsix = no more tsix expression
X chrom inactivation = not random anymore = only happens on chrom where removed tsix = remove so xist always turned on
X lacking tsix alwayas inactivated - skewed choice
Describe what “choice” of which x to inactivate is chosen by - generally
Shift in balance of xist and tsix expression
Tsix = neg regulator of xist
Describe what “choice” of which x to inactivate is chosen by - undifferentiated
In both directions xist and tsix
Xist and tsix bi allelic
Describe what “choice” of which x to inactivate is chosen by - onset of inactivation
Somehow restricts expression of one
On inactive x = xist wins
On active x = tsix transcript wins
Monoallelic expression corresponding to future inactivated and active x
Describe tsix in males
ONLY TSIX EXPRESSED= never detect xist on single male chrom
Name some of the factors which regulate xist
Trans acting - autosomal =
Feed into xist regulation
Known pluripotency transcription factors, REPRESS xist - expression of core group tfs
Oct4, sox2 nano, prdm14, rex1
What is rex1, compared to rnf12
Rex 1 = negative regulator xist
Regulated by enzyme rnf12 - by differentiation - regulated at level of protein stability
Describe rnf12 specifically
Enzyme that causes degradation of rex1 protein - through ub proteasome system = and is encoded on X
When differentiation happens = rnf12 upregulated = degrade rex1 = promote xist transcription
Does rnf12 differentiation switch work in males
NOOOO
Since there is only one x so not enough rnf12 expressed to get rid of rex1 and allow xist expression
Rex1 =comes from autosomes but rnf12 on x chrom
In females= lots rnf12 produced = more effective at triggering degradation rex1, so xist on
Describe a potential mechanism for counting
The rex1 /rnf12 system
= ensuring proper ration fo autosomes to active x
Maybe - female cells can adjust xist expression in relation of autosome to x, diff from male cells
Describe amount of rex1 when NO x chrom inactivation
Lots of rex1, less xist (keeps levels of xist low)
Undifferentiated es cells
Describe amount of rex1 when x chrom inactivation
Rex1 degraded by rnf12, more xist
= x chrom inactivation
Tsix is transcribed antisense to Xist, and its transcription is correlated with Xist repression. What experiment could you do to determine if this is because of Tsix transcription or the Tsix RNA molecule? - ideas
Inject tsix only and see if rna does something - first ko tsix
Or make mutation in sequence
Can take out promoter then insert gene somewhere and see
Inject micrornas to degrade rna
Is it the rna or act of transcription?
- Design an experiment that would deplete the RNA without preventing transcription
(Stop transcription at xist locus and maybe prevent tsix but not xist - see if lack of transcription has effect) - RNAi? Would also knock down Xist!! (Rnai - technique, not strand selective so would degrade both tsix and xist)
- Express Tsix from another genomic location and cripple the endogenous promoter
- Transcription terminator placed in such a way that Xist expression is not affected could also work
Describe xist and its repeat sequences
Multi tasking x linked transcript
Repeat sequences in xist rna are binding sites for important regulators -regions of repeat with short motifs
Name specifically what segments xist responsible for what
Repeat A= part of xist important For silencing, forms transcriptional repression complex
Repeat C= related to poly comb protein - prc1 recruitment - has have in x chrom inactivation
Repeat E= ability of xist to localize to zx chrom, localization
Name 2 general approaches for defining functions of xist rna
Genetics
Biochemistry
Describe general approaches for defining functions of xist rna - BIOCHEM
Identify proteins that interact with xist rna or parts of it - found likely pieces, what proteins binding to these pieces rna, that are important for these functions
Put pieces of rna into cell extracts and see what Biden to them
Describe general approaches for defining functions of xist rna - GENETICS GEN
Delete parts of rna and ask how xist function altered, could apply to other non coding rnas and proteins
Study full lengths, take away pats and see what happens = lead to discovery of repeats A,C and E and their function
Describe general approaches for defining functions of xist rna - GENETICS DELETE A REPEAT
No repulsion fo transcription but xist can still localize to x chrom
Describe general approaches for defining functions of xist rna - GENETICS DELETE C REPEAT
No prc association or h3k27me on inactive x but repression can still happen
Describe general approaches for defining functions of xist rna - GENETICS DELETE E REPEAT
No xist localization to Inactive x, repression compromised
DESCRIbe what happens when remove E repeat of xist - gen
Remove repeat or proteins that bind to it = leads to dispersal in the nucleus
Xist spread, all over nucleus, not localized to inactive x anymore
Usually foci in cells =binds to inactive x, other proteins = distributed all over cell
DESCRIbe what happens when remove E repeat of xist - specific binding
Identified by biochemistry
Hnrnp u (saf-a) is required for xist coating of inactive x
Has separate rna and dna binding domains that tether xist rna to inactive x, also binds to nuclear scaffold surrounding the nucleus (reason why inactive x moves to periphery) - anchors xist to chrom it comes from
What happens when remove c repeat of xist - gen
Prevents recruitment of prc1 and h3k27me to inactive x - associated with area around inactivated x -put on by poly comb protein
What happens when remove c repeat of xist - specific, roles
Inactive x enriched in h3k27me
Prc1 and prc2 complexes localize to inactive x when xist expression unregulated
Localization xist dependent
Prc1/prc2 seem to have a role in stabilizing the silent state of the inactive x but are not absolutely required for silencing
What happens when remove A repeat of xist
Compromises silencing function of xist and recruits spen and other chromatin proteins
Can still localzi,e but not silence
Compromise a ability of xist to create heterochromatin on inactive x
What does spen do - remove A repeat of xist
Spen associated with hdac3 - maybe has something to with Histone deacetylation
Idea = rna methylation - modifies rna itself, important for action of xist
Can x chrom inactivation be maintained w/o xist - explain
Yes can be maintained in absence xist expression
One formed in early embryo = stably propagated without xist - more of a property of regular heterochromatin
What is needed for stable maintained of x chrom inactivation
Stable maintenance of x chrom inactivation in the embryo requires dna meth (by dnmt1a nd dnmt3b) and hypoacteylation (low levels acetyaltion seeen on inactive x, but also happens for reg heterochromatin) of histones
Describe reactivation of inactive x
Occurs in primordial germ cells - parallel with erasure of imprinting
Naturally during dev - global epigenetic remodeling - removed imprinting and x chrom inactiavtion
Describe reactivation of inactive x - why is it important
Important for reprogramming somatic cells to stem cells - IPscs
Describe induced pluripotent stem cells
Lots fo progress, take differentiated somatic cells and reprogram cells back to stem cells
Important for regenerative medicine
Could replace cell types in adults if injury
Inactive x poses a barrier - especially with female cells
What are the types of inducted pluripotent stem cells based on
3 types based on state of x chrom inactiveation
Reprogramming efficacy depends on how well we can repgram inactive x - get it back to undifferentiated blastocyst stage
What happens when failure of x chrom reactivation - for induced pluripotent stem cells
Seems to corrrelate with lower pluripotency in reprogrammed cells
Impetus to develop strategies to reactive inactive x
Implications for x linked genetic disorders
Name and describe the 3 types of induced pluripotent stem cells
Class 1 - Xaxa = no xist, mouse like rare , reactive x - differentiate the best, more effectively
Class 2 - Xaxi = xist still on, common, do not differenatiate as well, inactive x still around
Class 3 - xaxe, xist off, common, partial reactivation = xist off, differentiation works better
What is unique to inactived x chrom- Maintenance
CGI methylation at promoters is common on inactive x - as compared to rest of genome, methylation of cpg islands - leaded to stable and maintenance happens later in x inactiavtion
Occurs at later stages of x chrom inactivation
