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X-Inactivation

X-Inactivation. Rana Hussein Part 1, September 2010. Outline. What is X- inactivation How is it mediated and for what purpose Assessing X-inactivation by cytogenetic techniques Assessment of X-inactivation by molecular genetics techniques Skewed inactivation and disease.

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X-Inactivation

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  1. X-Inactivation Rana Hussein Part 1, September 2010

  2. Outline • What is X- inactivation • How is it mediated and for what purpose • Assessing X-inactivation by cytogenetic techniques • Assessment of X-inactivation by molecular genetics techniques • Skewed inactivation and disease

  3. What is X–inactivation and what’s its purpose? • Lyonisation is the process by which the genes on one of the two X chromosomes in a female are switched off through transcriptional silencing. • It is thought to be random – either maternal or paternal are inactivated per cell. • It takes place in female somatic cells of the early embryo (and some specific hematopoietic progenitors) as germ line cells require the activity of both X-chromosomes. • This results in females being a mosaic of cells – a proportion with an inactive paternal X and a proportion with an inactive maternal X chromosome. • Genes in the pseudoautosomal region are not inactivated

  4. How is it mediated? • The control of X-inactivation is dependant on two mechanisms • Counting mechanism • Choosing mechanism • There are several genes that are associated with this process located in a region of the X chromosome referred to as the X inactivation Centre (Xic) at Xq13.2

  5. The Counting Mechanism: • This involves the cell assessing the number of X chromosomes per diploid set • There are several models proposed for this mechanism • Blocking factor – proposed that a cell is able to produce enough blocking factor to coat only one X-chromosome • Transient homologous pairing of the two X chromosomes – deletion of two genes in the Xic region (Tsix and Xite) stops this pairing from taking place. Annual Review of Genetics; 2008, 42:733 – 772

  6. The Choosing Mechanism: • This involves designating one of the X chromosomes as the active chromosome and the other(s) as inactive • This has been proposed to be a multi step process including non-coding RNAs and regulatory factors (both in cis and trans to the inactive X chromosome) • Xist (X inactive specific transcript) gene • this gene is thought to initiate X-inactivation (initiation) • produces a non- coding RNA • This is transcribed from Xi • RNA coats Xi thereby changing it’s chromatin environment rendering it inactive • Not all genes are inactivated and it’s cover of Xi isn’t continuous Annual Review of Genetics; 2008, 42:733 – 772

  7. The Choosing Mechanism contd: • Tsix gene • Name is reversed as it’s product is the antisense transcript of the Xist gene. • It’s function is to regulate the production of the Xist RNA by repressing it’s activity • Regulatory switch that determines whether the X in cis to it is turned on or off by determining whether Xist is turned on (Xi) or off (Xa) • It does this by recruiting chromatin modifiers to the promoter of Xist • Tsix regulated by DXPas34 and Xite (non-coding RNA) Annual Review of Genetics; 2008, 42:733 – 772

  8. DXPas34 and Xite • These carry out their function by enhancing the activity of Tsix • They are themselves also regulated • DXPas34 is regulated by the chromatin insulator protein Ctcf and it’s binding partner and transcriptional activator Yy1. This complex both induces Tsix transcription and may also block the access of Xist to downstream transcription enhancers – by doing this it would play a role in choosing which X remains as the Active X. • These 2 enhancers (CTCF and YY1) also bind to the Xist promoter – mutations in the CTCF binding site tend to show skewing of X-inactivation towards preferential inactivation of the mutant X. • Other factors that play a role in X chromosome inactivation inhibition include Annual Review of Genetics; 2008, 42:733 – 772

  9. Maintenance of X-inactivation and Methylation • Maintenance: ensuring that the inactive state of the chosen X is maintained through every subsequent cell division • This occurs in all cells except the germline cells (reactivation) • DNA methylation and histone hypoacetylation are associated with the inactive X and seems to be an important part of maintenance of the inactive state. • DNA methylation at the Xist promoter of the active X chromosome seems to play an important role in suppressing Xist in the active X chromosome

  10. Assessing X-inactivation by cytogenetic techniques • Karyotyping - to indicate the loss of a chromosome or gain of one or to assess for abnormalities in the chromosome • This can be determined by culturing of lymphocytes and examining them at mitotic metaphase for their banding patterns • These would not only indicate the loss of the X chromosome but can also indicate any abnormalities in the X chromosome • To assess X inactivation status, further tests can be carried out by FISH using a probe for the Xist locus to determine presence or absence of the Xist locus. • It’s absence would indicate an abnormal amount of products from X-linked genes that can have adverse effects on individuals

  11. Assessment of X-inactivation by molecular genetics techniques • Chemiluminescence • This is the emission of light as the result of a chemical reaction • Involves the digestion of patient DNA by EcoRI and EagI (methylation sensitive enzyme) • This is followed by running the digests out on a gel, hybridisation with a chemiluminescent probe and blotting • The degree of hybridisation of the probe to either the normal allele or the expanded allele can be an indication of X-inactivation

  12. Skewed inactivation and disease • The mechanism of X-linked in activation as previously mentioned compensates for females having 2 X chromosomes. • If a female is heterozygous for a certain condition, preferential silencing of the mutant allele renders her a carrier of the mutation • There tends to also be a growth disadvantage of active mutant X chromosomes during proliferation • The product of the X-linked gene plays a part in the manifestation of the disease • In those diseases that have a circulating gene product, a heterozygous female would show an averaging out of the product in their blood (such as in the case of haemophilia). • If the X inactivation is skewed and thereby results in greater silencing of the normal allele, the female can be seen to be a manifesting heterozygote.

  13. Case study • Female patient referred to Cardiff for Fragile X. On testing by Triplet repeat PCR, patient found to have 1 normal allele. • Further testing by chemiluminescence showed the patient with a normal and expanded allele. • Clinician phoned to ask if there was even stronger evidence from our results that explained why this patient is a manifesting heterozygote.

  14. Comparison of fluorescence of normal and expanded allele indicates greater fluorescence of expanded allele, indicating skewed X-inactivation

  15. Assessment of Skewed X-inactivation by Cytogenetic techniques • The Androgen Receptor gene on the X-chromosome • This test relies on the size of the repeat on the two X-chromosomes being of different sizes, on the methylation status of the x-chromosome and the methylation-sensitive restriction enzyme HpaII • A CCGG repeat found at a site close to the AR site has variable methylation depending on whether the X-chromosome is active (unmethylated) or inactive (methylated). • The methylation-sensitive enzyme HPaII will digest the active (unmethylated) X-chromosome but not the inactive (methylated) X. • Two PCRs are carried out – one with undigested patient DNA and the other with digested patient DNA – any digested DNA will fail to amplify • Comparison of peak heights in both samples for both alleles will indicate whether or not X-inactivation is skewed. • If X-inactivation is skewed towards one allele then there will be a greater loss of peak height or complete loss of peak height seen in this allele compared to the other.

  16. Skewed X inactivation and autoimmunity • Skewed X activation also implicated in autoimmune disease. • Several hypotheses including: • The loss of mosaicism hypothesis • The reactivation hypothesis • The haploinsufficiency hypothesis

  17. References • Medical Genetics, Connor and Ferguson-Smith • Human Molecular Genetics, Strachan and Read • Agrelo, R. and Wutz, A., X-inactivation and disease, Seminars in Cell & Developmental Biology, 21 (2010): 194-200 • Penny, GD. Et al, Requirement for Xist in X-chromosome inactivation, Nature; 1996, 379: 131-137 • Payer, B. and Lee, JT., X chromosome dosage Compensation: How mammals keep the balance, Annual Review of Genetics; 2008, 42:733 – 772 • Willard, HF. And Carrel L., Making sense and anti-sense of the X inactivation centre, PNAS, 2001, 98(18): 10025-10027 • Wolff et al., Small marker X chromosomes lack the X inactivation centre: implications of karyotype/phenotype correlations, Am. J. Hum. Gen., 1994, 55(1):87-95 • Linert et al., The X chromosome in immune functions: when a chromosome makes the difference, Nature Reviews Immunology, 2010, 10: 594-604 • Barakat et al., X-changing information on X-inactivation, Experimental Cell Research, 2010, 316: 679-687 • Royce-Tolland et al., The A repeat links ASF/SF2- dependent Xist RNA processing with random choice during X-inactivation, Nature Structural and Molecular Biology, 2010, 17(8): 948-955

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