21. X-inactivation in females

Question 1

What is the purpose of X-inactivation?

How does it occur?

Answer 1

Mechanism for gene dosage compensation

Equalises X chromosome gene expression between males and females

Random transcriptional silencing of either maternal or paternal X chromosome

Question 2

When does X inactivation occur?

Answer 2

As cells differentiate in late blastula stage

Pattern of X inactivation inherited stably in daughter cells

Question 3

Describe the structure of the inactive X

Answer 3

Highly condensed, heterochromatic state

Visualised as a Barr body

Question 4

Describe the mechanism of X inactivation

Answer 4

Initiated at XIC

XIST is a lncRNA transcribed from inactive X only - acts in cis

XIST spreads from XIC, recruits polycomb proteins, coats X chr –> DNA methylation and histone acetylation

Question 5

What is the role of Tsix?

Answer 5

Transcribed from antisense strand of XIST - represses XIST expression through DNA methylation and histone acetylation

Question 6

How do XIST and Tsix regulate inactivation?

Answer 6

Prior to inactivation, both Xs weakly express XIST and Tsix

At inactivation, future inactive X ceases to produce Tsix and XIST expression increases

XIST is repressed on active X by methylation of promoter

Question 7

What are PARs?

Answer 7

Pseudo-autosomal regions - contain genes present on both X and Y

Escape inactivation as dosage compensation not required

Question 8

What is the effect of X:autosome translocations on X inactivation?

Answer 8

Translocated part of X escapes activation

Or is XIC is translocated, autosome can be inactivated

Ideally, in a balanced t(X:A), the intact X is inactivated (so that autosome isn’t)

Question 9

What is the effect of random X inactivation on XLD disorders?

Answer 9

Trait is less severe in females as 50% of cells with pathogenic X are inactivated

Female more severely affected due to non-random/skewed X inactivation due to variants in Xist or Tsix

Question 10

What is replication banding?

Answer 10

Used to differentiate between active and inactive X

BrdU added - incorporates U during replication, A-U cleaved by UV light –> no G-banding

Inactive X is late replicating so no banding pattern. Normal banding pattern on active X

Question 11

How can skewed X in activation be tested for?

Answer 11

Most commonly HUMARA assay

gDNA digested with methylation-specific enzyme - digests unmethylated allele only

Amplification of CAG STR in AR gene - only methylated allele is amplified