2003 Silver Fleece Awards

1. 2003 Silver Fleece Awards • The recipients represent “an egregious example of people feeding a line of bull to the public.” 1. Clonaid- Claim to have cloned humans • Says that cloning will “enable mankind to reach eternal life” • Claims that memories and personality will be transferred into a brand new body 2. Urbana Nutrition, Inc. - • Market “Longevity” as anti-aging “There are no methods or products that actually slow, stop or reverse aging” Leonard Hayflick, U. of California, San Francisco Source- AP news, Friday, March 14, 2003

2. X chromosome inactivation-Review- Panning an Jaenisch, RNA and the epigenetic regulation of X chromosome inactivation. Cell 93:305, 1998 A. Introduction- • Barr bodies first described in females in 1949 • Turners syndrome (45,X) are Barr body negative; Kleinfelters syndrome (47, XXY) are Barr body positive • Lyon hypothesis- one of the two X chromosomes in female is inactivated; all but one is inactivated if multiple X chromosomes - referred to as “dosage compensation”

3. X chromosome inactivation • Introduction- • X-chromosome inactivation occurs at day 3 of embyrogenesis • Inactivation process is random • Inactivation state maintained throughout life • A few genes remain active in the inactive X chromosome, including XIST at Xq13

4. Dosage compensation comparisons 1X 1X 2X 2X 2-fold increase in males 2-fold decrease in females Stably inactivate one X chromosome

5. X chromosome inactivation in flys and worms • Distinct mechanisms to achieve dosage compensation • C. elegans- Dosage compensation by reducing gene activity by two fold on each X chromosome • Mechanism- if one X-, XO-lethal gene is on resulting in male determination • Drosophila- Stimulate X gene transcription 2-fold in males to equal levels from each X • chromosomes in female

6. Mammals-X-inactivation is used to compensate for 2 X chromosomes Three steps- 1. choice- occurs in embryonic cells • Xist is expressed from both X chromosomes in female • Xist encodes 15 kb polyadenylated untranslated RNA that is unstable • Xist is gene located within Xic

8. Xist RNA expressed by both X chromosomes Xist Xist Xist RNA interacts with stabilizing factors Xist Blocking factor prevents Xist RNA stablization and spreading Mechanism of Xist-mediated silencing Inactive Active

9. Inactivated X chromosome Xist RNA

10. X-inactivation- Step 2 2. initiation- begins at X-inactivation center (Xic) • Xist RNA spreads in cis to coat chromosome • Note that Xist does not interact directly with DNA, but likely through a protein intermediate • Xist gene on other X chromosome is silenced

11. X-inactivation- Step 3 3. Spread- propagated bidirectionally from Xic • Xist methylation required for silencing of Xist • Dnmt KO- Male X and two female X are all inactivated because Xist gene remains on and Xist RNA coats chromosome

12. 13 p 12 11 12 13 14 q 21 24 mouse autosome X-inactivation- observations • Xist is necessary and sufficient for X inactivation (using 450kb YAC) • insert Xist transgene on autosome results in inactivated autosome

13. X-inactivation- observations • but Xist is neither necessary or sufficient to maintain X inactivation in somatic cell hybrids • Thus initiation and maintenance of X inactivation are likely distinct mechanisms • Xist maintains inactive state in cis, not trans

14. Xist Gene Random inactivation Blocking factors Preferential inactivation Prevent inactivation delete X-inactivation Mechanism in mammals • If mutate Xist promoter- preferential X inactivation on chromosome with mutation • possibly due to failure to compete with blocking factor • Delete Xist exons 1-5- mutant chromosome chosen but not inactivated

15. Mechanism in mammals In Extraembryonic tissues, paternal X is always inactivated • if paternally inherited mutant X – observe no X inactivation in extraembryonic tissue in females • if maternally inherited mutant X - WT phenotype in extraembryonic tissue (i.e .WT X always inactive)

16. X-inactivation X-controlling element (Xce) mapped to a 6 kb region in Xist gene and is required for X inactivation • Model- Mutually exclusive binding of blocking factor to Xce on one X, and of initiator factor to Xist on other X • Marsupials and in mice extraembyonic tissues- paternal X always inactivated in females Reason???

17. X-inactivation • Xist RNA may cause replication origins to fire late, resulting in heterochromatin formation • modulate histone acetetylation • The Drosophila mof gene is required for dosage compensation and is an acetyltransferase

18. Clerc and Avner, Science 290:1518, 2000 X-inactivation is reprogrammed during development Random X inactivation in extraembryonic tissue! Thus, an epigenetic, non-erased tagging must occur normally with male X.

19. Science 295:345, 2002

20. Recall CTCF is involved in genomic imprinting • Recent information- • The factor CTCF may also be involved in X-chromosome choosing. Science 295:345, 2002 CTCF binds and activates XIST anti-sense transcription (called Tsix), which prevents Xist expression, which keeps that X chromosome active

21. Identifiction of the X-inactivation region (X