The MCDB MentorsClub Present

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The MCDB Mentors/Club Present Dr. Larry Gold
The Choices We Make and Where They Take Us 
From Academic Science to Biotech to
Therapeutics and Diagnostics for Health
Care April 27,7 -800 pm A2B70 Pizza and
Soda Reception Coffee and dessert 800- 830
pm, Learning Lounge B0056
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• The Final Exam (May 6 1030 AM, here)
• will be worth 70 or 75 points to make up for lost
  points along the way.
• Review Session May 5, 230-4 PM B121
• You still get to drop one quiz, one problem set,
  one paper discussion, and two class periods of
  clicker points.

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Dosage compensation
Female vs. male
Most organisms have 2 copies of each autosome
2 copies of X
1 copy of X
If the amount of transcript produced from each
chromosome is important, then there must be
some compensation mechanism that allows both XX
and X to be viable genotypes
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Dosage compensation fits into the sex
determination pathways for invertebrates
C. elegans Sex determination pathway
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C. elegans
XA ratio determines activation of dosage
compensation genes that direct the level of
expression from X
Transcription from X chromosome is decreased by
1/2 in hermaphrodites
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Drosophila is the opposite of C. elegans
transcription from the X is increased 2x in males
male
female
No Dosage Comp.
Dosage Comp.
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Summary of dosage compensation in Drosophila and
C. elegans
Drosophila compensates by increasing
transcription from the X in males (1 X
animals) XA 1.0, Sex lethal on Female sex
determination and dosage compensation XA 0.5,
Sex lethal off Male sex determination and
dosage compensation MSL-1, 3, MLE, roX
RNAs Assemble on the single X chromosome in
males and allow twice as much product to be
transcribed
C. elegans compensates by decreasing
transcription from the Xs in females (2 X
animals) XA 0.5, XO-lethal on male sex
determination and dosage compensation XA
1.0, XO-lethal off hermaphrodite sex
determination and dosage compensation sdc-3,
dpy-26, dpy-27, dpy-28 (dcd genes) Assemble on
both X chromosomes and downregulate (by half)
transcription from each X chromosome
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• Which of the following is true of a worm that is
  homozygous mutant for xol-1 (lf)--one of the
  dosage compensation genes?
• XX embryos develop into male worms
• XO embryos develop into hermaphrodite worms
• XO embryos die due to lack of X chromosome gene
  products
• XX embryos die due to overproduction of X
  chromosome gene products

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• In which chromosomal combination would a loss of
  function mutation in the Sxl (sex lethal) gene be
  lethal? (.5)
• XX
• XY
• Neither
• Both

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Inactivation is random and takes place at about
the 32-cell stage
In mammals dosage compensation is accomplished
by inactivating (almost) the entire X chromosome
G, 5.22
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The Barr body represents the inactive X
chromosome
Cell from XX female
Cell from XXX female
G, 5.21
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X inactivation in action Calico cats
All female mammals are mosaics for genes on the
X chromosome, illustrated here by coat color. O
(orange, dominant) and o (black, recessive) are
two alleles on the X chromosome for coat color.
G, 5.22
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• (.5) You are doing chromosomal analysis of an
  individual with two X chromosomes who appears
  externally male. You find that one of the X
  chromosomes contains a large translocation from
  the Y chromosome.
• If you were able to look at multiple tissue
  samples from this individuals gonad, you would
  expect to see
• The gonadal cells have all differentiated into
  testis tissue
• The gonadal cells have all differentiated into
  ovarian tissue
• The gonadal cells have not differentiated into
  testis or ovarian tissue
• Some gonadal cells have differentiated into
  testis, some into ovarian tissue

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• The entire X chromosome is not inactivatedthere
  is a small section where transcription still
  takes place
• This has consequences for individuals with
  abnormal sex chromosome combinations
• XXY infertile
• XO infertile
• But XXX normal

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Review How the structure of chromatin affects
transcription
Trancription ON
Transcription OFF
Euchromatin --loose, easy access by trx
factors --increased acetylation of
histones --decreased methylation
Heterochromatin --tight, difficult access by trx
factors --decreased acetylation of
histones --increased methylation
? inactivation methylation
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Methylation

• Accomplished by methylases and methyl
  transferases
• Occurs on a cytosine (C) followed by a G

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Differential methylation of promoters controls
the transcriptional state of genes
The methylation state of a gene can be changed
during development
5.18
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A complex on the X chromosome, the XIC is
responsible for X inactivation
Xist
-- does not get translated into a protein --
instead, makes a long RNA that coats the
inactive X chromosome, forming an Xist-chromatin
complex How does it work?
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The state of Xist is opposite to the rest of the
X chromosome
When Xist promoter is ON (ACTIVE,
unmethylated), Xist is made, and that X
chromosome is inactivated
When Xist promoter is OFF (INACTIVE, methylated),
Xist is not made, and that X chromosome is
active

• Further proof that Xist is responsible for
  inactivation
• Xist RNA localizes to the inactive X chromosome
• Xist expression precedes overt X inactivation
• If Xist is mutated, both X chromosomes remain
  active
• If Xist is placed onto an autosome, the
  autosome
• becomes inactive

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Once Xist is activated on one of the X
chromosomes, additional methylation events take
care of further inactivating the genes on that X
chromosome
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Xist is initially transcribed on both X
chromosomes, then restricted to only the inactive
X
Before inactivation
during
after
Xist rna photo
Two Xlinked transcripts Red Pgk mRNA Blue
Xist mRNA
G, 5.23
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How does this worK? Xist is trancribed from
different promoters at different times
P0
P1
P2

• Initial transcription of Xist is from P0 promoter
  on both Xs
• this RNA is unstable and degrades.
• Subsequent transcription from P1/P2 promoter
  occurs only on one of the Xs
• this RNA is stable
• on the other X, Xist is methylated in this
  way, the activation of one X is maintained
• Xist RNA then spreads along the inactive X,
  presumably blocking transcription from that
  chromosome.

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• Suppose that an XX individual is heterozygous for
  a loss of function mutation in Xist. What result
  would you expect regarding X inactivation?
• Both X chromosomes are active
• Both X chromosomes are inactive
• X inactivation occurs non-randomly, on the
  chromosome with the Xist mutation
• X inactivation occurs non-randomly, on the wild
  type chromosome
• There is no effect on the inactivation of the X
  chromosomes

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Regulation of Xist how is the inactive X chosen?
Positive regulation a competence factor that
helps turn on transcription of stable Xist RNA
to initiate inactivation ? (postulated)
Negative regulation two known factors

• 1. Tsix an antisense transcript exactly
  complementary to Xist, found on the opposite
  strand
• Tsix is expressed at high levels from the
  active X chromosome and at low levels from the
  inactive (the opposite of Xist).
• Blocks Xist?
• 2. Blocking factor when bound, blocks access to
  Xist enhancer, Xist not transcribed

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How does a cell count the number of X chromosomes?

• In XO and XY individuals, the X is not
  inactivated

• In XXY and XX individuals, a single X is
  inactivated

• In XXX individuals, 2 Xs are inactivated

Probably cells are not actually counting the Xs,
but rather protecting only one X chromosome from
being inactivated (blocking factor must be on
autosome)