X chromosome Inactivation Flashcards
(95 cards)
1
Q
What happens to the X chromosome in Drosophila?
A
Males increase transcription from the X chromosome: 2 fold
2
Q
What happens to the X chromosome in C elegans?
A
Females decrease transcription from the X chromosome: 2 fold
3
Q
What happens to the X chromosome in Mammals?
A
Females completely inactive one of the X chromosome
This chromosome is referred to as a Barr body
4
Q
What is special about the X chromosome?
A
Males and females need to balance the gene expression from the X chromosome
5
Q
What is a Barr body?
A
A silenced, transcriptionally inactive X chromosome was first identified as a Barr body
Xa : active X chromosome
Xi: inactive X chromosome
6
Q
What is special about Xi?
A
Xi is adjacent to the nuclear envelope
On Xi all CpG islands on Xi are methylated, histones are hypoacetylated, no transcription is observed
7
Q
Which X chromosome is inactivated?
A
One X chromosome in each cell is randomly inactivated early during embryonal development
The silencing of the Xi is inherited through multiple rounds of divisions
One or the other
8
Q
What are females?
A
Female mammals are X mosaic (different cells of the body one or the other X chromosome is silent)
9
Q
What happens in a cell culture?
A
In cell cultures an Xi stays inactive; reverts at a frequency of about 10^-8
Reversal of the Barr body is incredibly rare
10
Q
Explain Molecular events during early embryo development in female fetuses (Drawing to help explain)C
A
1) We have a zygote, with a partially pre-inactivated X chromosome, and a maternal X chromosome that is imprinted –> The Xm is active and the Xp is silenced in the embryo
2) Two cell stage: Repeat silencing of Xp
3) Four cell stage
4) Mourla: Genic silencing of Xp, and progressive Xp silencing
5) Blastocyst: Continuation of Xp inactivation in the placenta only (extra embryo tissues), Xm is active in placenta, Xp is silenced in placenta
There is reactivation of Xp in the embryonal tissues, Xp is reactivated in the developing embryo, both Xp and Xm are now active in the embryo, so random inactivation of Xp or Xm takes place in the embryo only
6) In the developing fetus, the embryo becomes X mosaic (Xp or Xm is active, only one is active not both)
7) The placenta is maternally imprinted (Only Xm is active)
8) In the developing germ line cells (future reproductive cells), there is pre-meiotic reactivation of both Xp or Xm takes place –> The “activation” imprint is placed on XIST of both X chromosomes
11
Q
What happens in mature females?
A
They are mosaic, which means some cells have an active maternal X chromosome and some cells have an active paternal X chromosome
11
Q
What happens at the start of development in females?
A
Paternal X chromsome is inactive at the start of the development and is activated later on alongside the maternal X chromosome
12
Q
Why is X chromosome inactivation so important?
A
Very significant epigenetic component in the context of development
13
Q
What is Rett syndrome?
A
Progressive neurodevelopment disorder
It is X linked, and affects methylated DNA binding protein
14
Q
What are the symptoms of Rett syndrome?
A
One of the causes of mental retardation in females
Normal development seen for 6-18 months, then gradually lose speech, seizures, autism, hand writing behaviour, then stabilize and patients usually survive into adulthood
Affected males are extremely rare, which means males will die early in development
15
Q
How is Rett syndrome caused?
A
Caused by a X linked mutation
The mutation is lethal in hemizygous XY males
In XX females, X chromsome inactivation and mosaicism leads to variability affected heterozygotes (Half of the X’s could be silenced)
70-80% of Rett cases are caused by mutations in an X linked gene encoding MeCP2 (Methyl- CpG- Binding Protein 2)
Epigenetic disease
Pathological mechanism is not clear, but linked to mosaicism
16
Q
Explain the MECP2 model for Rett disease
A
Mecp2-/- mutant mice: Model for Rett syndrome
In these mice, the induced expression of MeCP2 in late stages of development reversed Rett syndrome
17
Q
Explain the establishment of Xi
A
Silencing initiates from a specific position on the X chromosome and this position is called XIC (X chromosome inactivation centre)
18
Q
What is the importance of XIC?
A
XIC is needed for X silencing/inactivation
19
Q
What does X: Chr22 and X: Chr14 translocations lead to?
A
These lead to the inactivation of the whole hybrid chromosome is certain regions of theX chromosome is present
The other part of X is not silenced
20
Q
What does XIC contain?
A
XIC contains three critical elements:
1) Xce (X controlling element): controls random inactivation of XIST
2) Xist (X inactivation specific transcript) gene
3) Tsix (antisense transcript to Xist) gene
XIC contains two opposing transcripts (transcribed in opposite directions): Xist and Tsix
21
Q
Explain XIST and TSiX
A
They are lncRNAs
Tsix is the anti-sense transcript of XIST
Xist inactivates the X chromosomes from which it is expressed
Tsix is involved in the random inactivation of Xist
Xist inactivates the X chromosomes from which it is expressed
Tsix is involved in the random inactivation of Xist
22
Q
What are the proposed roles of XIST?
A
Proposed roles of XIST:
1) Formation of silent nuclear compartment
2) Gene silencing via the repeat A
3) Mediator of chromosome interactions (TADs)
4) Recruitment of Chromatin Modifiers
23
Q
What happens when Xist is expressed?
A
Precludes the expression of Tsix
Don’t know exact mechanism
24
What does XIC look like?
Draw locus
25
What controls Xist expression? (There is a picture to help explain this concept)
Tsix
In early embryonal development both Tsix and Xist are transcribed
Tsix is transcribed in the opposite direction of Xist
Possibly over runs the promoter of Xist (mopping of promoters in FLO11)
Deletion of Tsix promoter does not preclude the repression of X chromosomes, but it is not random
26
How does the cell randomly choose one X chromosome?
It is believed that Tsix plays a major role in this process
Tsix expression is stimulated by Oct4/Sox2/Nanog/C-myc, the genes that maintain stem cell phenotypes in embryonic stem cells
During differentiation the expression of Oct4/Sox2/Nanog/C-myc decreases, Tsix expression also goes down
It is possible that the two Tsix promoters compete in "all or none" fashion until one "loses" and allows for the unhindered expression of Xist
The possibility of a special TAD formation has also been proposed
27
What is a proposed mechanism for the Xist preclusion?
A TAD could be involved in the decision on which X chromosome to inactivate
There is an active loop and an inactive loop
In the human, Chic1, cdx, Tsix are in the inactive/supressed loop, and Xist, JPX, Fxt, Slc16A2 are in the active loop
Important to note: Tsix promoter is repressed, so inactive loop, and Xist promoter is active and expressed, so in active gene
28
What are the roles of Xist?
The role of the expressed Xist lncRNA is better characterized
Xist gene encodes a 19kb polyA + untranslated RNA transcript
Xist transcript is expressed at high levels from Xi but not from Xa
Large amounts of Xist wrap up the Xi and the histone hypoacetylation and DNA methylation of the entire chromosome follow
On Xa, XIST transcription is silenced by methylating Xic DNA
29
What is Xist necessary for? What is Xist not necessary for?
Xist is necessary for initiation of inactivation in cis
Xist is not needed for the maintenance of the inactive X chromosome
Once established, the heterochromatin on Xi is very stable
30
Explain Xist RNA during XX female ES cell differentiation
Researchers used RNA FISH
In undifferentiated ES cells: Xist is expressed from both X chromosomes, there is no accumulation of Xist, and there is no obvious heterochromatin and euchromatin in undifferentiated cells
Differentiation Initiated: One X chromosome shows a lot of Xist expressed, this is going to be inactivated the other X chromosome shows little Xist expressed, will remain active
Fully differentiated: The X chromosome that showed a lot of Xist expressed, is now inactivated and formed into a Barr body, Xist is still expressed in large amounts
Distinct euchromatin and heterochromatin domains in differentiated cells
31
Explain the cascade of events that follow the selection of one of the X chromosomes
1) Xist
2) Xist RNA covers the whole chromosome, and chromosome is wrapped by Xist transcript
3) Heterochromatin gets built up onto the chromosome on top of the Xist RNA
When Xist is expressed, the Xist transcript wraps around the X chromosome
This initiates a cascade of heterochromatin formation to repress this X chromosome
Xist associates with selected loci throughout the X chromosome
Xist wraps Xi during early stages of X-inactivation
4) Xist recruits PRC1, the initiatior of gene silencing (PRC1's role is to recruit PRC2)
5) Recruitment of histone modifier, PRC2, then H3K27 methyl transferase follows
Overall, chromosome is wrapped with Xist and then recruit PRC1
32
How is Xist recruited and how does it bind? There is an image to help explain this
There are focal points along the chromosome that recruit Xist
Don't know exact mechanism
Xist binds to DNA
33
Explain how the Xist loci associates with PRC2
Xist loci on the inactivated X chromosome overlaps with the clustering of PRC2 (H3-K27 Methyl transferase)
Initation of histone methylation followed by demethylation and formation of heterochromatin
34
What drives the inactivation of the Xp chromosome in the zygote?
The repressive imprint on Xm (maternal) XIC locus is imposed during oogenesis in females
The imprint is de-methylated at CpG islands at the promoter of Tsix so that Tsix is expressed from the Xm but not Xp
The imprint is not established on the Xp during spermatogenesis
In the zygote the Xist RNA is expressed by the Xp chromosome and not by the Xm chromosome and not by the Xm chromosome
Later on (in the epiblast) Xp is reactivated
35
What does the imprint tell the cell?
The imprint tells the cell that a X chromosome is maternal is a CpG island that is demethylated
When Tsix is expressed, then Xist is suppressed
36
What is an IAP? Where is it found?
LTR Transposon, which is found in human and mice genomes
37
What is a PGC?
Progenitor Germ line cells
38
Explain Germline Epigenetic re-programming in mice
In the zygote, there is global demethylation, active and passive in paternal and passive in maternal
In the blastocyst, there is global re-methylation, but imprints are not erased, then in the primordial gremlin cells, imprints are erased, and then imprints are re-established in male and female, and there is global re-methylation
39
Explain imprints during development
Imprints are imposed in germline cells before birth in males
Imprints are imposed in germline cells postnatally in females
There is a massive de-methylation and re-methylation during development
Imprints are also erased in primordial germ cells, and then re-establishd
Imprints survive global demethylation that occurs during differentiation
There is erasure of marks between zygote and blastocyst in males, they come back and then erased at primordial
Female maintain marks until primordial, where they are erased and re-built
39
Explain what happens during development at the Blastocyst stage
At the blastocyst stage, whole genome is demethylated. Blastocyst marks the beginning of differentiation of somatic cell (kind of like starting with a clean state)
Then, there is global remethylation (whole genome is re-methylated and there are histone modifications)
40
Explain what happens during development in the primordial germ cells
At the primordial germ cells, everything is demethylated, so that epigenetic patterns can be re-established, which includes imprinting
40
Explain what happens during development in the primordial germ cells
At the primordial germ cells, everything is demethylated, so that epigenetic patterns can be re-established, which includes imprinting
Imprinting establishment in male germ line vs female germ line have different timing, as discussed above
After primordial germ cells, there is global re-methylation again
41
Explain histones in the maternal and paternal genome
Maternal genome has histones but little to no methylation
Paternal genome has no histones, but lots of DNA methylation
42
What is the difference between male and female imprints?
Male imprints: push for competition and fertility
Female imprints: push for survival
43
Explain what happens to the epigenetic marks at each stage
There is a massive demethylation and re-methylation during development
Between the Zygote and Blastocyst stage, epigenetic marks are erased in the male genome
At the primordial germ line cell stage, all epigenetic marks are erased to re-establish the epigenetic marks
44
What is the mechanism for how parental imprinting occurs at other loci?
An allele from one parent is "imprinted" by DNA methylation during germ line development
These epigenetic marks at the imprinted locus are transmitted through germ line cells and persist through the massive demethylation of DNA in the very early embryonal stages of development
In all somatic cels of the organism this parentally imprinted gene remains methylated and repressed throughout the life of the organism
The allele from the other parent is active in all cells, which is why only the gene from one of the parents is expressed
In certain cells, only maternal or paternal allele will be expressed in certain cells
This process involves CTCF and Cohesion
45
What is the cycle of imprinting in progeny (There is a picture to help explain this process)
Maternally and paternally DNA methylated ICRs gain DNA methylation in oocytes and sperm, respectively
Imprints are maintained despite reprogramming and global changes in DNA methylation after fertilization
Imprinting involves the establishment of heritable TADs in the ICR
CTCF and cohesion are central in the formation of TADs
Binding of CTCF to DNA is regulated by DNA methylation and depends on methylation
46
What happens in the progenitor germ line cells during imprinting?
DNA methylation of both maternal and paternal alleles is erased
Imprints are re-established during gametogenesis in a sex-specific fashion before being transmitted to the next generation
47
How is CTCF binding to DNA regulated by DNA methylation?
Binding of CTCF depends on methylation
Sequence specific DNA binding protein prefers to bind to a specific sequence that contains cytosine
When cytosine is methylated, CTCF cannot bind to the sequence
Allows for regulation of the epigenetic landscape of the cell
48
What are the imprinted gene clusters of most importance?
Igf2r, Pws, Igf2, Dlk1
49
What are the imprinted gene clusters?
Imprints and ICRs are clustered in 15 genome regions
These clusters contain 160 genes and multiple non-coding RNAs
50
What is Igfr2?
Regulates growth of embryo/Regulates growth of whole organism
Mother under expresses this
Insulin like growth factor 2 receptor
51
What is Pws?
Involved with epigenetic disorders
52
What is Igf2?
Insulin growth factor 2
Regulating growth of the whole organism
53
What is Dlk1?
This determines behaviour of adult, which suggests that behaviour is determined through embryonic development/imprinting
54
Where are Igf2 and H19 located?
Occupy nearby loci on chromosome 11
55
What is H19?
Non-coding RNA
56
What happens to Igf2 and H19 in any diploid somatic cell?
Only the paternal Igf2 is expressed (maternal Igf2 is always silenced)
Only the maternal H19 is expressed (paternal H19 is always silenced)
57
What is the mechanism for how Igf2 and H19 are expressed/silenced?
Sex specific DNA methylation patterns on imprinted loci, including IRCR, are established in gametogenesis, and inherited with the gamete, and can escarole cytosine methylation, plus erasure/resetting in early development
Differential methylation pattern remains for duration of organisms life
58
What happened in the Dnmt1+/+ mutation?
DNA methylation of paternal H19 allele, which silences/prevents parental H19 from being expressed
Suggests need Dnmt1 to methylate and impose imprints
59
Why is allele specific DNA methylation so important?
For the maintenance of the imprinted state
60
What happened in the Dnmt1 -/- mutation?
Remove DNA methylation of paternal allele, loss of Dnmt1 and now both paternal and maternal H19 are ON, and both paternal and maternal Igf2 OFF
61
How are H19 and Igf2 controlled on the paternal allele? There is a diagram to help illustrate this
H19 and Igf2 are controlled by ICR
The ICR is a dual chromatin boundary/silencer element
Methylation of ICR spreads over H19 and turns it OFF --> this is the silencer activity of ICR
Methylation of ICR shuts off promoter of paternal H19
Methylated ICR does not bind CTCF
In the absence of CTC, a chromatin boundary is not formed and Igf2 is exposed to a distant enhancer
62
How are H19 and Igf2 controlled on the maternal allele? There is a diagram to help illustrate this
ICR is not methylated, so CTCF protein binds to ICR and a loop is formed excluding Igf2 from the TAD of the enhancers
The enhancer now activates H19
63
How are loops formed by CTCF? There is a diagram to help illustrate this
Besides ICR, there are two additional CTCF sites in the locus
When ICR is not methylated, CTCF does not bind and a different loop is formed, now positioning Igf2 and the ehancer in a TAD
64
How are chromatin contacts established between regulatory elements and the maternal alleles at the Igf2/H19 locus? There is a diagram to help explain this
TAD formation: H19 forms a TAD with an enhancer
Igf2 forms a chromatin loop inaccessible by the enhancer
DMR: differentially methylated region
Nuclear Matrix: associated domain, holds the shape of chromatin, and acts like a scaffold
CTCF and Cohesion play critical roles in these structures
65
How is methylation and binding of CTCF important in the paternal allele? There is a diagram to illustrate this
Methylation of ICR on parental locus does not allow binding of CTC, which means promoter of H19 is inactive (Inactive part of loop)
A loop is formed by the two distal CTCFs bringing Igf2 to the enhancer, and now Igf2 is active and brought to the enhancers, making the active part of the loop
the DNA of the ICR is methylated, which prevents CTCF binding, so the insulator function at this site is abrogated, and so downstream insulator contacts insulator upstream of Igf2 and "pulls" the enhancer which activates Igf2 gene
66
How is methylation and binding of CTCF important in the maternal allele? There is a diagram to illustrate this
Absence of methylation of ICR allows binding of CTCF, so H19's promoter is active and close to enhancers, active part of the loop.
Igf2 remains a loop away from the enhancer, because no TAD formed with Igf2 due to H19 activity, so Igf2 is part of the inactive loop, and this loop gets heterochromatinized
Only a single maternally derived allele shows H19 gene activity, which is induced by downstream enhancers
67
What does the binding of CTCF cause?
Causes CTCF to dimerize with CTCF bound to Igf2 promoter, excluding it from TAD with the enhancers
68
IWhat happens in Beckwith- Wiedemann Syndrome?
DNA methylation at ICR in both paternal and maternal alleles, which means there is a high dosage and over-expression of Igf2
Igf2 is not repressed (lack of repression)
No H19
Significant amount of growth factors expressed due to lack of repression, so overgrowth syndrome
Infants are larger than normal
Specific body parts grow abnormally large
69
What happens in Silver Russel Syndrome?
No DNA methylation at ICR, so Igf2 is not expressed in paternal and maternal and instead H19 is over-expressed in both maternal and paternal,
Igf2 is repressed, while H19 is over expressed, so slow growth, smaller size of specific body parts, normal head size
70
What is the Callipyge phenotype example?
Sheep have more muscle, and this phenotype is highly desirable in sheep breeding
Phenotype is due to constant muscle twitching, making muscles appear bigger
Imprinting of a locus encoding several proteins and miRNA
Trait is inherited from father
Locus contains 4 imprinted genes (DLK1, PEG11, GTL2, and MEG8)
71
What is the mechanism for the Callipyge phenotype? There is a diagram to explain this process
GENE DOSAGE!!!
Over-expression of PEG11 is what causes deficiency, and can be inherited from both the mother and the father
Inherited from only mother: Normal
Inherited from only father: Sheep will be normal
If inherited from both mother and father , dosage compensation, so sheep will be normal
It is the non coding RNAs expressed from these genes that lead to the phenotype
72
What happens in the Callipyge locus?
CLPG is inherited from the father
DLK1 and PEG11 are over expressed, GTL2 and MEG8 are under expressed
73
What happens in the non callipyge locus?
CLPG inherited from mother, so DLK1 and PEG11 are underexpressed, GTL2 and MEG8 are over expressed in females
74
What is the callipyge locus in humans?
14q32 which contains several maternally or paternally imprinted genes
Disregulation causes developmental disorders (TS and KOS)
miRNAs expressed from this locus are driving muscle growth
75
How is the 14q32 locus regulated in humans? There is a diagram to help explain
In maternal MEG8 is methylated, and the methylation of this locus means nothing will be expressed downstream
In paternal MEG8 is unmethylated
Non methylation drives expression
76
What is the "Tug of War" Hypothesis?
Mother and Father have different strategies to maximize their genetic contributions to future generations
The "race" to gain the evolutionary advantage leads to this genome competition
Offspring is believed to be nourished from maternal tissue
Fathers want to maximize the competitiveness of their progeny, while females want progeny to survive, hence the "tug of war" between maternal and paternal gene expression
77
What does the Parent Offspring Hypothesis suggest?
Maternal genome strategy: Partition resources to herself, all current and future progeny (in which she has an equal genetic stake)
Paternal genome strategy: Maximize resource allocated to its true embryo (current progeny) at the expense of the mother and future siblings
78
What are the predictions of the parent offspring conflict hypothesis?
Imprinted genes are involved in fetal growth or nutrient acquisition and will reflect the selfish strategy of mothers and fathers, which can be seen by Igf2 being only paternally expressed, and Igf2r being expressed by the maternal allele, which is an insulin like growth factor 2 repressor (silences Insulin like growth factor 2)
79
What evidence supports the Parent Offspring Conflict Hypothesis?
Igf2 mutant mice are 40% smaller than wild type (mother wins)
Igf2r mutant mice are oversized (father wins, but child dies)
Igf2, Igf2r double mutant is normal (the child wins, but the mom and dad loose)
80
What is the additional support to suggest the Tug of War Hypothesis?
Many paternally imprinted genes act as embryonal promoters of growth (Igf2, Peg1, Peg3, Rasgrf1, Dlk1)
Many maternally imprinted genes act as embryonal suppressors of growth (Igf2r, Gnas, Cdkn1C, H19, Grb10)
Egg laying mammals and non mammals do not have genome imprinting
In the callipyge locus, the paternal strategy seems to work only in the muscle (overgrown tissue)
81
How do TADS and insulated neighbourhoods work?
Enhancers and promoters are brought together through a cohesion CTCF loop
Dysfunctional looping by this mechanism can lead to various types of cancers and other disease phenotypes, which leads to altered CTCF binding sites and/or improper looping leading to abnormal expression
82
What is the hierarchy of a chromosome structure?
Chromosome territories, TADS, and insulated neighbourhoods
83
What is a chromosome anchor? There is a diagram to help explain
CTCF bound site interacting with another CTC bound cohesion ring
84
What evidence is there to suggest the insulation for an insulated neighbourhood? There is a diagram to help explain
Deletion of insulated neighbourhood anchor leads to gene misregulation
Mutations of insulated neighbourhood anchors in tumour cells lead to oncogene activation
85
What is the model for DNA looping by a mediator and cohesion?
There is single DNA loop, but multiple enhancers may be bound at the same time, generating multiple loops
OCT4, SOX2, NANOG bind the mediator, which binds RNA polymerase II at the core promoter, thus forming a loop between the enhancer and core promoter
The transcription activator bound to the mediator binds to the cohesion loading factor, NIPBL, which provides a means to load cohesion
Both the mediator and cohesion are necessary for normal gene activity
86
What is the model of a TAD that consists of nested insulated neighbourhoods? (There is a diagram to help explain this)
Methylation of CTCF binding sites also influence the type of loop formed by the interaction between CTCF and cohesion
CTCF binding is regulated by DNA methylation, so cannot bind to methylated DNA
87
What is the model of a TAD that consists of two insulated neighbourhoods nested within a TAD spanning CTCF-CTCF loop?
There is a diagram to explain this
88
What is the model of a TAD that consists of an insulated neighbourhood
There is a diagram to explain this
89
What is the structure of the human beta globin locus?l
Human beta globin locus comprises of five coding genes that are specifically expressed in erythroid cells:
The embryonic specific, epsilon the fetal restricted: Gy and Ay and the adult expressed, delta and beta globin genes
Expression of all these genes require a strong upstream enhancer known as the LCR
90
What is the LCR?
Encompasses five DNAaseI hypersensitive sites (HS1, HS2, HS3, HS4, HS5)
LCR is in physical proximity to the relevant beta type globin gene promoters in a developmental stage specific manner
Switch from fetal to adult glob gene expression normally occurs around the time of birth
LCR regulates gene expression and is a mega-enhancer
91
What is the HBBP1?
Region mediates dynamic chromatin contacts with the 3' HS1/HS5 and epsilon globin at fetal stages and adult stages, respectively
92
What is the model of chromatin configurations of the human beta globin locus in fetal and adult erythroblasts?
This makes most sense to have in a diagram
