2003 Silver Fleece Awards 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 The recipients represent “an egregious example of people feeding a line of bull to the public.” Source- AP news, Friday, March 14, 2003

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”

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 X chromosome inactivation

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

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

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

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

Xist RNA Inactivated X chromosome

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

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 X-inactivation- Step 3

 Xist is necessary and sufficient for X inactivation (using 450kb YAC)  insert Xist transgene on autosome results in inactivated autosome X-inactivation- observations mouse autosome p q

 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 X-inactivation- observations

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

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) Mechanism in mammals In Extraembryonic tissues, paternal X is always inactivated

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???

 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 X-inactivation

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

Science 295:345, 2002

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 Recall CTCF is involved in genomic imprinting

Identifiction of the X-inactivation region (X