18.01.21 X-inactivation Flashcards Preview

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Flashcards in 18.01.21 X-inactivation Deck (21)
1

What is the purpose of X inactivation?

X-inactivation provides a mechanism of gene dosage compensation equalising X chromosome gene expression from males and females. Failure to induce inactivation in the embryo or in extraembryonic tissues is usually lethal.

X inactivation is an epigenetic process resulting in transcriptional silencing of one X chromosome in females, and therefore in both sexes having only one functional copy of X.

2

Where is X inactivation initiated?

X inactivation is initiated at the X inactivation centre (XIC) on Xq13.

Original evidence for the existence and location of an XIC came from studies of X:autosome translocations (t(X;A)) and del(X) karyotypes, which interfered with manifestation of inactivation.

3

What is the role of XIST in X inactivation?

The gene XIST (X-inactive-specific transcript) is a long non-coding (lnc) RNA molecule transcribed only from the inactive X. It acts, in cis, on the chromosome from which it was transcribed.

The Xist RNA molecule spreads along the chromatin outwards from the XIC, along with the Polycomb proteins it recruits. Resulting heterochromatin modifications such as histone deacetylation and methylation of CpG islands are epigenetic processes which organise the chromatin into a closed confirmation.

Xist is required to initiate inactivation but not to maintain it – proof from investigations with transformed cell lines with deletion of Xist – X inactivation pattern is maintained.

4

Summarise the process of X inactivation.

1. At blastocyst stage: Xist RNA is expressed in an unstable form and postulated blocking factors prevent Xist upregulation and/or its association with the chromosome in cis.

2. XistRNA becomes upregulated through stabilisation, transcriptional upregulation or release of the blocking factor. LINE might participate in the spreading process in some way - either through assoication with nucleprotein complexes including Xist or by a mechanism such as repeat-induced gene silencing

3. Stabilised Xist RNA coats the X chromosome before its inactivation,

4. Transcriptional silencing of genes on the X chromosome occurs as a result of Xist RNA coating and is rapidly followed by a shift to asynchronous replication timining of the X chromosome

5. Chromatin modifications, such as the histone deacetylation and methylation of promoters of X-linked genes, as well as the recruitment of the histone variant macroH2A, presumably transform the Xist RNA-coated chromosome into a stably inactive and condensed chromatin state.

5

What is the role of LINEs in X-inactivation?

Long Interspersed Nuclear Elements (LINEs) have been implicated in X inactivation as these regions are commonly found in the X chr but depleted in regions escaping inactivation. LINEs are also found on autosomes and may help to explain how inactivation can spread into an autosome in t(X;A).

Chow et al, 2010, propose that expression from transcriptionally active LINE elements may contribute to spreading of X inactivation into regions that are more resistant to silencing.

6

What is the role of Tsix?

The Tsix antisense transcript is transcribed from the antisense strand of XIST gene. It is expressed in the very early embryo before random inactivation and is involved in repressing expression of XIST.

Tsix is thought to regulates chromatin structure by altering histone tail modifications and DNA methylation at the XIST promoter.

7

What are XIST and Tsix RNA expressed?

Prior to inactivation both X chromosomes weakly express XIST and Tsix RNA. At the onset of inactivation, the future inactive X ceases to express Tsix RNA and increases XIST expression. The future active X continues to express Tsix for several days.

Soon after the coating of the inactive X with Xist transcript, there is rapid loss of its transcriptional machinery (RNA Pol II, TAF10 and TBP proteins).

8

How is inactivation of XIST and Tsix maintained?

Inactivation is maintained by high levels of DNA methylation, low levels of histone acetylation on the inactive X and accumulation of novel histone variant called macroH2A

Once inactivation has occurred on one X, repression of the other XIST allele is maintained by methylation of its promoter.

Inactivation is re-set during oogenesis and both Xs are active. Having both active may be necessary to permit recombination in meiosis (not possible if one is condensed).

9

What is XACT?

Recent evidence suggests there is a lncRNA that coats the active X chromosome – known as X-active coating transcript (XACT). In female human embryonic stem cells in which XIST is repressed, XACT is expressed from and coats both X chromosomes, and this correlates with significant reactivation of the inactive X chromosome.

Expression of XACT appears to be specific for pluripotent cells as its expression decreases during differentiation (Vallot et al, 2013).

10

Which regions escape X inactivation?

PAR1, PAR2
non pseudo-autosomal XY homologous region on Xq21.3 and Yp11.2 (~4Mb).
Estimated that 15% of X-linked genes escape inactivation

11

What is the evidence that there is some kind of X counting mechanism in place?

In an abnormal karyotype with additional X chromosomes, the additional Xs will all be inactivated, but some genes will still be transcribed, resulting in functional trisomy (or more).

12

Which X chromosome is preferentially inactivated in a BALANCED t(X:A)?

The intact X is preferentially inactivated, and the two derivative chromosomes will together comprise the functional X.

However, gene disruption at the translocation breakpoint may complicate a phenotype.

It is possible that there is no ‘preferential inactivation’; rather selection of cells with the preferred inactivation pattern, after random inactivation has occurred.

13

Which X chromosome is preferentially inactivated in a UNBALANCED t(X:A)?

In an unbalanced X chromosome (translocation or other chromosome abnormality) there would ideally be selection for cells with the inactivation pattern which resulted in the least imbalance.

There is no hard and fast rule for which X should be inactivated in a karyotype with an X chromosome imbalance, however as XIC is relatively close to the centromere (and therefore less likely to be included in the translocated segment), it is more likely that the abnormal X should be inactivated.

14

What is skewed X-inactivation?

Skewed X chromosome inactivation is considered to occur at a ratio of >75%; extreme skewing at >90%. Skewing of X inactivation is known to increase with age.

15

What are the possible effects of skewed X-inactivation?

Random X inactivation means an X-linked dominant trait is usually less severe in a female carrier than in a male. Theoretically, in 50% of cells the X containing the pathogenic variant is inactivated, and the normal allele is transcribed from the active X chromosome.

16

Which factors may influence the level of X-inactivation skewing?

Age

A female heterozygote may show skewed X inactivation resulting in preferential inactivation of the X containing the pathogenic variant.

Skewing may also result from selection due to a growth or other advantage at the molecular level for cells with inactivation of the mutated X.

However, if there is no preferential inactivation, statistically one X may be inactivated more frequently than the other. Therefore, chance could lead to a female heterozygote for an X-linked trait showing a phenotype, if there is no selection at the cellular level.

The extent of skewing may be different within different tissue types, therefore blood may not always be representative if used for X-inactivation studies.

17

What techniques can be used to investigate skewed X-inactivation?

Replication banding
Methylation-specific PCR
HUMARA assay

18

What is replication banding and how is it performed?

A cytogenetic technique to determine which X is inactivated, for example in t(X;A). Analysis of sufficient metaphases can allow calculation of a ratio of any skewed inactivation.

1. Addition of BrdU to a culture causes incorporation of uracil during DNA replication.

2. The inactive X is late replicating, whereas the active X replicates as usual.

3. Addition of BrdU mid way through the cell cycle causes some DNA to have A-U (single H bond) and some to have A-T (two H bonds).

4. Subsequent UV treatment preferentially cleaves A-U bonds (single H bond).

5. SSC elutes away broken fragments resulting in ssDNA where uracil has been incorporated.

6. Giemsa stain only binds to dsDNA, not ssDNA facilitating visual differentiation between early and late replicating chromatin.

19

How are the results of replication banding for X-inactivation interpreted?

Inactive X shows no banding pattern, as entire chromosome is late replicating.

Active X retains banding pattern, as some parts replicate late and some parts early – therefore some areas incorporate T and some uracil, resulting in a banding pattern.

It can also be possible to visualise whether inactivation has spread into an autosomal segment of a der(X)t(X;A), if the autosomal segment is large enough to easily detect.

20

How can MS-PCR be used to assay X-inactivation skewing?

DNA is chemically modified using sodium bisulphite; unmethylated cytosine are deaminated to produce uracil (read as thymidine during PCR).

This enables the design of PCR primers specific to the methylated and unmethylated alleles.

The ratio between maternal and paternal inactive Xs can be calculated.

21

How can the HUMARA assay be used to determine X-inactivation skewing?

Most commonly used technique to determine X-inactivation patterns.

This is a PCR-based technique which amplifies a polymorphic CAG repeat region located within the first exon of the androgen receptor gene.

Genomic DNA is first digested using HpaII, a methylation-specific enzyme that will only cut the unmethylated (i.e. active) allele therefore allowing us to distinguish between the active and inactive X chromosomes.

PCR primers are specific to regions flanking HpaII sites, such that digestion of the unmethylated allele disrupts PCR amplification. As a result, only the methylated (i.e. inactive) allele will give rise to a PCR product.

PCR products are then separated by capillary electrophoresis and amplification patterns between the undigested and digested alleles can be compared