2003 Silver Fleece Awards 1. Clonaid- Claim to have cloned humans –Says that cloning will...

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 increasein males

2-fold decreasein females

Stably inactivate one Xchromosome

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

Xist

Xist

Blocking factor prevents Xist RNA stablization and spreading

Active

Inactive

Xist RNA interacts with stabilizing factors

Xist RNA expressedby 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

11

12

13

21

24

12

13

14

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-inactivationX-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