Download Prof. Kamakaka`s Lecture 15 Notes

Eukaryotic chromosomes
Bacterial
DNA is in a nucleoid body
There is one large DNA molecule
Circular
Eukaryotic
DNA is in chromosomes
There are many molecules
Linear
The DNA in the diploid nucleus is ~2 meters long.
It is present in a nucleus that is a 1000 cubic microns.
Function of chromosomes
Packaging
Regulation
Total human DNA is 3x109 bp
Smallest human chromosome is 5x107 bp
The DNA in this chromosome is 14 mm long
The chromosome is 2um long
7000 fold packaging!
1
Amount of DNA varies between species
Amount of DNA varies in eukaryotes
Salamander genomes are 20 times larger than human genomes
Barley genome is 10 times larger than the rice genome
Barley and rice are related.
Measurements of DNA length
Amount of DNA/nucleus = C value
Species
DNA content (pg or 10-12g)
haploid
Sponge
Drosophila
Human
Lungfish
Locust
Frog
Yeast
0.05
0.2
3.5
102
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4.2
0.03
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C-Value paradox
This is often called the C value paradox.
There is no phylogenetic relationship to DNA content
There are sibling amphibian species - they look morphologically
identical but have 4-fold difference in DNA content
How do we account for the differences in DNA content/nucleus
No of genes
Gene size
Distance between genes
3
Junk DNA
1)
Number of genes could vary in these organisms
Lungfish would have to have 30 fold more genes than humans
Barley and rice have the same number of genes but vastly
different DNA contents.
Number of genes does not correlate with amount of DNA in a
cell.
2)
Size of genes could increase as genomes increase
Drosophila genome is 30 times larger than E.coli
Average coding region of a gene is 1-2 kb long in Drosophila
E. Coli genes are only slightly shorter
Drosophila genes are not 30 times larger than E. coli genes.
Introns and promoters etc increase the size to some extent but
cannot account for all of the increase.
3)
Amount of DNA between genes increases
Humans= 25,000 genes. Size of human genome is 3x109 bp
Yeast= 6000 genes. Size of yeast genome is 1.4x107 bp
The DNA between genes (intergenic region) varies.
A large fraction of intergenic DNA is repetitive
Nearly 60% of the human genome is repetitive.
Less than 5% of the yeast genome is repetitive.
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Repetitive DNA
5
Interphase nucleus
Heterochromatin
Inactive genes
Constitutive heterochromatin
Repetitive DNA
(Telomere, centromere etc)
Gene poor
Transcriptionally silent
Euchromatin
Gene Rich
Active genes
Facultative heterochromatin
Gene rich
Transcriptionally silent
6
Epigenetics and development
1.
2.
Differentiation
De-differentiation
2n DNA content
same DNA content
> 200 cell types
Cloning by nuclear transfer --> regenerate entire organism from
transfer of single nucleus (e.g. Dolly)
Induced pluripotent stem cells (iPS) --> expression of 4 genes are
sufficient to transform differentiated cells to “stem” cells

Both processes must involve reprogramming of epigenome!
Epigenetics: Gene regulation through stable activation/repression
Heritable changes in gene activity that cannot be explained by
changes in gene sequences
This is essential for normal cell differentiation and development
of an organism after fertilization
Epigenetics imposes restrictions to the plasticity of totipotent
embryonic cells
During early development there is a progressive restriction of
cellular plasticity accompanied by acquisition of cell type
specific patterns of modifications on genes
Epigenetic modifications impose a cellular memory that
accompanies and enables stable differentiation
xxxxx
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Epigenetic inheritance during mitosis
sperm
egg
Embryo
All Genes are poised for activity
Cell commitment
Specific genes activated
All other genes inactivated
Active genes maintain activity
Inactive genes remain silent
Active genes maintain activity
Inactive genes remain silent
10
Epigenetics and gene ACTIVATION during development
Heritable changes in gene expression that do not involve
changes in DNA sequences
All Genes not active in all cells
. examples:
•Developmentally regulated / tissue specific gene expression
. mechanisms:
•Presence of Transcription factors
Active Inactive
Transcription activator
+++
---
Gene activation
•examples:
•Developmentally regulated / tissue
specific gene expression
•X chromosome dosage compensation
•Gene Imprinting
•Position effect variegation (PEV)
Cell/tissue specific transcriptional activators bind to enhancers
of genes that have binding sites for these factors
-Aid in recruitment of enzymes that modify chromatin at the
promoter
- Aid in recruitment of the general transcription machinery
and RNA polymerase
Promoter TATA Inr
Gene
Enhancer
The enhancer functions to activate genes. There are specific
sequences that bind TISSUE SPECIFIC transcription factors.
The binding of these factors induces gene activation 100 fold!
Active gene promoters have DNA sequences (CpG residues)12
that are not methylated and are bound by specific
transcription activators.
Cell specific Activation
Different Enhancers bind different
tissue and cell specific transcription
activator proteins and this enables
specific gene activation in specific cells
HNF3
Liver Cell
Liver
gene1
Brain
gene1
HNF3
Liver
gene2
Brain
gene2
Brain Cell
NZF2
Liver
gene1
Brain
gene
1
NZF2
Liver
gene2
Brain
gene
2
13
Epigenetics and gene REPRESSION during development
Heritable changes in gene expression that do not involve
changes in DNA sequences
All Genes not active in all cells
. examples:
•Developmentally regulated / tissue specific gene expression
. mechanisms:
•Absence of transcription activators
•Presence of repressors
• Changes in Chromatin
• Changes in DNA methylation
Active Inactive
Transcription activator
+++
---
Repressor proteins
---
+++
Specific Histone Modi
+++
---
Specific Histone Modi
---
+++
DNA methylation
---
+++
sperm
egg
Embryo
All Genes are poised for activity
Cell commitment
Specific genes activated
All other genes inactivated
Active genes maintain activity
Inactive genes remain silent
Active genes maintain activity
Inactive genes remain silent
mechanisms:
• Changes in Transcription factors
• Changes in DNA methylation
• Changes in Chromatin structure
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Cell specific Repression
Different Enhancers bind different
tissue and cell specific transcription
activator proteins and this enables
specific gene activation in specific cells
HNF3
Liver Cell
Liver
gene1
Brain
gene1
HNF3
Liver
gene2
Brain
gene2
Brain Cell
NZF2
Liver
gene1
Brain
gene
1
NZF2
Liver
gene2
Brain
gene
2
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Gene Silencing and its importance
In any given cell, only a small percentage of all genes are
expressed
Vast majority of the genome has to be shut down or silenced
Knowing which genes to keep on and which ones to silence is
critical for a cell to survive and proliferate normally during
development and differentiation
Transcription factors bind active genes and keep them active
DNA methylation of inactive genes keeps them inactive
Cell commitment
Specific genes activated
All other genes inactivated
Active genes maintain activity
Inactive genes remain silent
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Inactive chromatin
Heterochromatin
Inactive
Euchromatin
Active
Constitutive heterochromatin: Repetitive DNA-Centromeres,
telomeres etc
• Repetitive DNA tends to recombine expanding/contracting
repeats. Preventing repetitive DNA from recombination is
critical for cell survival
•Constitutes ~ 20 % of nuclear DNA
• Highly compacted,
• Always transcriptionally/Recombinationally inert
Euchromatin + facultative heterochromatin:
• constitutes ~ 80% of nuclear DNA
• less condensed, rich in genes,
•Euchromatin is transcriptionally active
• the rest is transcriptionally inactive (but can be activated
in certain tissues or developmental stages)
These inactive regions are known as “facultative
heterochromatin”
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Facultative heterochromatin
Regions of genome, rich in genes that are condensed in specific
cell types or during specific stages of development.
It includes genes that are highly active at a particular stage of
development but then are stably repressed.
X-chromosome inactivation in vertebrates (Dosage compensation)
No. of transcripts are proportional to no. of gene copies
Diploid- 2 copies of a gene
Genes on X-chromosomes
In females there are two copies of a gene. In males there is
one copy.
XX
2
XY
1
Measuring transcript levels for genes on the X chromosome in
female and male show that they are equivalent.
Dosage imbalance is corrected!
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Dosage compensation
•examples:
•Developmentally regulated / tissue
specific gene expression
•X chromosome dosage compensation
•Gene Imprinting
•Position effect variegation (PEV)
In Drosophila in the males there is an increase in transcription
from the single X chromosome. A inhibitor of transcription is
turned off in males allowing for full expression from the one X
chromosome
In nematodes there is a decrease in transcription from both
X chromosomes- protein binds the 2X chromo and causes
chromosome condensation which reduces transcription.
In mammals, X chromosome inactivation occurs in females by
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formation of heterochromatin on one X chromosome
Mammalian X-chromosome inactivation
Mammalian males and females have one and two X chromosomes
respectively.
One would expect that X-linked genes should produce twice as
much gene product in females compared to males. Yet when one
measures gene product from X-linked genes in males and females
they are equivalent.
This phenomenon, known as dosage compensation,
X chromosome inactivation in females is the mechanism behind
dosage compensation.
In females, one of the X chromosomes in each cell is inactivated.
This is observed cytologically. One of the X-chromosomes in
females appears highly condensed. This inactivated chromosome is
packaged into heterochromatin and forms a structure called a
Barr-body.
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Dosage compensation
Dosage compensation in mammalian females occurs by shutting
off of most of the genes on one X chromosome in females.
The inactive X chromosome becomes heterochromatic.
It is called a Barr body
XCI is random.
It occurs at the 500 cell stage of the embryo
For a given cell in a developing organism, probability of the
maternally or paternally derived X being inactivated is equal.
Once inactivated, it is stably propagated so that all the
thousands or millions of cells descended from that embryonic
cell maintain the same chromosome in the Heterochromatic
state.
Xist is ON - Xist RNA coats the X- X chr is OFF
Tsix is on- Tsix pairs and inactivates Xist -X chr is ON
X chr with Xist gets methylated!!!!
Genes on methylated X chromosome are not active.
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XY
XX
reactivate
X
egg
X
sperm
XX
XX
Tsix Active
XX
Xist Active
Xist RNA
Inactivates
Xist RNA
Coat inactive X- methylate DNA
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CpG Methylation
Epigenetic mechanism #1: DNA methylation
• DNA methylation has long been correlated with repression of
gene expression
• DNA methylation mostly occurs on CpG dinucleotides and this
modification is only observed on inactive gene promoters
DNMTs
methyl group added
to the cytosine
methylation status is maintained
during replication/mitosis
Methylated DNA recruits methyl-CpG-binding repressor
proteins and other enzymes and this blocks access for RNA
polymerase blocking transcription
X-inactivation
The inactivation of one of the two X-chromosomes means that
males and females each have one active X chromosome per cell.
X-chromosome inactivation is random. For a given cell in the
developing organism there is an equal probability of the female or
the male derived X chromosome being inactivated.
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X-inactivation
zygote
Embryo
Inactivation
The embryo is a mosaic!
Once the decision is made in early development, then it is
stably inherited.
Patches of cells have the male X ON and patches of cells have
the female X ON
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This is a Developmental rule that overlays on top of Mendellian
rules!
Barr bodies
·
The inactive X-chromosome in normal females is called the
barr body
.
XXX females have 2 Barr Bodies leaving one active X
·
XXXX females have 3 Barr Bodies leaving one active X
·
XXY males have one Barr Body leaving one active X
(Klinefelter's syndrome)
·
X0 female have no Barr Bodies leaving one active X
(Turner's syndrome)
Given X-chromosome inactivation functions normally why are they
phenotypically abnormal?
Part of the explanation for the abnormal phenotypes is that the
entire X is not inactivated during Barr-Body formation (Escape loci)
Consequently an X0 individual is not genetically equivalent to an XX
individual.
XX female
XXX female
XY male
XXY27male
Mosaic expression
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
Xm
Xf
Xm
Xf
XmXf
XmXf
Xm
Xf
Xm
Xf
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Tortoise shell cats
Black
Orange
Enzyme O
The O gene is carried on the X chromosome.
Female cats heterozygous for the O gene on the X- chromosome
have a particular pattern called Tortoise shell.
According to Mendel’s rules the cats should be either orange or
black.
But the cats are neither! They are Tortoise shell.
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Tortoiseshell cats
All tortoiseshell cats are female
XY male
If normal OY gene is present on the X, the male is ginger
If mutant oY gene is present in male it is black
Female with O/O are ginger
Females with o/o are black
Females with O/o are tortoiseshell
In O/o females
X-chromosome inactivation happens at random
Some cells activate O gene making ginger pigment
Some cells activate o gene making black pigment
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Tortoise shell cats
According to Mendel’s rules these cats should be either orange
or black. But the cats are neither! They are Tortoise shell.
OO
x
oY
F1 females are Oo
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Tortoise shell cats
Female cats heterozygous for the O gene on the X- chromosome
have a particular pattern called Tortoise shell. According to
Mendel’s rules these cats should be either orange or black. But
the cats are neither! They are Tortoise shell.
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DNA Methylation is not perfectly inherited
during development/aging
Fraga et al., 2005
PNAS
102(30):106049.
xxxxxxxxx
34
Epigenetic inheritance and meiosis
sperm
egg
Embryo
Active gene from father maintains activity
Inactive gene from mother remain silent
35
Imprinting
Occurs on Autosomes
•examples:
•Developmentally regulated / tissue
specific gene expression
•X chromosome dosage compensation
•Gene Imprinting
•Position effect variegation (PEV)
Occurs only on some genes on autosomes
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Big bottom male x
normal true breeding female
203 big bottom:209 normal
C
N
N
N
C
50% N
Normal male
:
x
N
N
50%
big bottom female
N
N
C
N
100% normal
Calliphyge is Sex independent- both males and females can be big bottom
The callipyge gene is on autosome
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Big bottom is autosomal dominant?
CC
x
NN
100% Callipyge
NN
x
CC
0% Callipyge
Calliphyge gene is expressed when inherited from the males!!!
The calliphyge locus from mother is always silenced.
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Callipyge
Normal female X Normal male
The callipyge locus
from mother is
always silenced.
Normal phenotype
female allele is imprinted
(turned off) and male
allele is expressed
Normal female X mutant male
*
mutant female X Normal male
*
Mutant phenotype
Normal phenotype
Normal allele (from
mom) is imprinted
(turned off) and
mutant allele (from
dad) is expressed
Mutant allele (from
mom) is imprinted
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(turned off) and normal
allele (from dad) is
expressed
Imprinting
A small number of genes (~200) on autosomes
The allele from one parent is shut off.
In the egg/sperm, these genes are imprinted (turned off)
Imprinting leads to functional haploidy!
Gene is WT but no protein is made (i.e. mutant).
Abandoned safety net of diploidy.
Gamete
A=on
A=off
A=on
A=off
Somatic cell
40
The original imprint is erased in gametes and the new
imprint is established in progeny during gamete formation
Imprinted loci
41
DNA Methylation and imprinting
CTCF
Father
CH3 CH3
IGF2 Gene
Enhancer
CTCF
Mother
IGF2 Gene
Enhancer
War of the sexes
Why are perfectly good genes turned off?
Many maternally imprinted genes (inactive on the maternal
chromosome) are fetal growth factor genes
Tug of war
Father contributes active genes to enhance growth- extract
as many maternal resources for offspring as possible. He is
unlikely to mate again with that female. Advantage for
survival of his gene pool.
Mother silences these growth promoting genes to ration her
investment to any one offspring conserving resources for
future.
43
The phenotype is expressed only when the mutant allele is
inherited from the mother. Thus, mutant imprinted alleles can
remain masked when they are paternally inherited, but clinically
re-appear in one-half of children of carrier daughters
44