20.01.21 X inactivation in females

Question 1

What is X inactivation?

Answer 1

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

Question 2

What X is inactivated normally?

Answer 2

• Random inactivation of either the maternal or paternal X chromosome in each cell occurs as cells begin to differentiate (probably around the late blastula stage) - In subsequent mitoses this pattern of X inactivation is inherited stably by each daughter cell

Question 3

What does the inactivated X look like?

Answer 3

• highly condensed heterochromatic state and is visualised as a Barr body in the nucleus

Question 4

Where is X-inactivation initiated?

Answer 4

• X inactivation centre (XIC) on Xq13
• 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
• XIST is transcribed into an RNA molecule which stays in the nucleus and coats the X chromosome to initiate inactivation
• Inactive X becomes genetically inactive and replicates later in the cell cycle

Question 5

Briefly describe the main steps of X-inactivation

Answer 5

1) Before activation, Xist RNA is expressed in unstable form and the blocking factors prevent Xist upregulation
2) Xist RNA becomes upregulated through stabilization, transcriptional upregulation or release of blocking factors
- LINES (long interspersed nuclear elements) may be involved as they are found in regions that are inactivated but depleted in regions escaping inactivation
3) Stabilized Xist RNA spreads along the chromatin outwards from XIC - results in heterochromatin modifications such as histone deacetylation and methylation of CpG islands
4) Transcriptional silencing of genes on the X chromosomes occurs as a result of Xist RNA coating
5) Then get chromatin modifications and recruitment of the histone variant macroH2A that both transform the Xist RNA-coated chromosome into a stably inactive and condensed chromatin state

Question 6

Is Xist needed for initiation or maintenance of inactivation?

Answer 6

Xist is required to initiate inactivation but not to maintain it

Question 7

What is Tsix and what is its role?

Answer 7

• 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 regulate chromatin structure by altering histone tail modifications and DNA methylation at the XIST promoter - 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)

Question 8

How is inactivation maintained?

Answer 8

• 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

Question 9

What happens at oogenesis?

Answer 9

• 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)

Question 10

What region escape inactivation?

Answer 10

• Regions which are present on both X and Y (PAR regions) -‘Pseudo-autosomal regions’ 1) PAR1 - terminal 2.6Mb on Xp22.3, and a homologous region at the tip of Yp 2) PAR2 - terminal 320kb on Xq and Yq - Also estimated that ~15% of X-linked genes escape inactivation - If you have an abnormal karyotype with additional Xs - all of these additional Xs will be inactivated (but some individual genes may still be transcribed leading to trisomy for those genes)

Question 11

How are abnormal X chromosomes treated?

Answer 11

• X;autosome translocation - A problem arises as some of the X will be out of reach of Xist - In addition, if the XIC is within the translocated segment, inactivation can spread into parts of the autosome - Ideally 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 - 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.

Question 12

What is X inactivation skewing?

Answer 12

• 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

Question 13

What does random X inactivation mean?

Answer 13

• Should get random activation of either chromosome - So 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

Question 14

What are the phenotypic consequences of skewed X inactivation?

Answer 14

• A female HET may show skewed X inactivation resulting in preferential inactivation of the X containing the pathogenic variant
• HET variants in Xist or Tsix can cause non-random X inactivation
• 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 HET 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

Question 15

How can we detect X-inactivation?

Answer 15

1) Replication banding 2) Methylation-specific PCR 3) HUMARA assay

Question 16

Replication banding - method

Answer 16

• Cyto technique - Need to analyse enough metaphases to calculate ratio of any skewed inactivation
• Rough method:
  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
• Inactive X = no banding pattern (as entire chr is late replicating)
• Active X = retains banding pattern (as some parts replicate late and some early, so in some regions get incorporation of T and some U)
• 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

Question 17

Methylation-specific PCR - method

Answer 17

• 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

Question 18

HUMARA assay - method

Answer 18

• Most commonly used technique to determine X-inactivation patterns - PCR-based technique which amplifies a polymorphic CAG repeat region located within the first exon of the androgen receptor gene
• Rough method:
  1) Genomic DNA 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
  2) 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
  3) PCR products are then separated by capillary electrophoresis and amplification patterns between the undigested and digested alleles can be compared