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Eukaryotic chromosomes. Bacterial Eukaryotic DNA is in a nucleoid body DNA is in chromosomes There is one large DNA molecule There are many molecules Circular Linear. The DNA in the diploid nucleus is ~2 meters long. It is present in a nucleus that is a 1000 cubic microns.

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Eukaryotic chromosomes
Eukaryotic chromosomes

Bacterial Eukaryotic

DNA is in a nucleoid body DNA is in chromosomes

There is one large DNA molecule There are many molecules

Circular 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!


Amount of dna varies between species
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 0.05

Drosophila 0.2

Human 3.5

Lungfish 102

Locust 46

Frog 4.2

Yeast 0.03


C value paradox
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


Junk dna
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 3x109bp

  • Yeast= 6000 genes. Size of yeast genome is 1.4x107bp

  • 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.



Interphase nucleus
Interphase nucleus

Heterochromatin

Inactive genes

Euchromatin

Gene Rich

Active genes

Constitutive heterochromatin

Repetitive DNA

(Telomere, centromere etc)

Gene poor

Transcriptionally silent

Facultative heterochromatin

Gene rich

Transcriptionally silent


Epigenetics and development

2n DNA content

same DNA content,

> 200 cell types

De-differentiation

Differentiation

  • 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
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



Epigenetic inheritance during mitosis
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


Epigenetics and gene activation during development
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
Gene activation

TATA

Inr

Gene

  • 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

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) 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

Cell specific Activation

HNF3

Liver Cell

Liver gene1

Brain gene1

HNF3

Liver gene2

Brain gene2

Brain Cell

NZF2

Liver gene1

Brain gene1

NZF2

Liver gene2

Brain gene2


Epigenetics and gene repression during development
Epigenetics and gene REPRESSION during development transcription activator proteins and this enables specific gene activation in specific cells

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 transcription activator proteins and this enables specific gene activation in specific cells

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


Cell specific repression

Different Enhancers bind different tissue and cell specific transcription activator proteins and this enables specific gene activation in specific cells

Cell specific Repression

HNF3

Liver Cell

Liver gene1

Brain gene1

HNF3

Liver gene2

Brain gene2

Brain Cell

NZF2

Liver gene1

Brain gene1

NZF2

Liver gene2

Brain gene2


Gene silencing and its importance
Gene Silencing and its importance transcription activator proteins and this enables specific gene activation in specific cells

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


Inactive chromatin
Inactive chromatin transcription activator proteins and this enables specific gene activation in specific cells

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”


Facultative heterochromatin
Facultative heterochromatin transcription activator proteins and this enables specific gene activation in specific cells

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 XY

2 1

Measuring transcript levels for genes on the X chromosome in female and male show that they are equivalent.

Dosage imbalance is corrected!


Dosage compensation

  • examples: transcription activator proteins and this enables specific gene activation in specific cells

    • Developmentally regulated / tissue specific gene expression

    • X chromosome dosage compensation

    • Gene Imprinting

    • Position effect variegation (PEV)

Dosage compensation

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 formation of heterochromatin on one X chromosome


Mammalian x chromosome inactivation
Mammalian X-chromosome inactivation transcription activator proteins and this enables specific gene activation in specific cells

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.


Dosage compensation1
Dosage compensation transcription activator proteins and this enables specific gene activation in specific cells

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.


XY transcription activator proteins and this enables specific gene activation in specific cells

XX

reactivate

X

egg

X

sperm

XX

XX

XX

Tsix Active

Xist Active

Xist RNA

Inactivates

Xist RNA

Coat inactive X- methylate DNA


Cpg methylation
CpG Methylation transcription activator proteins and this enables specific gene activation in specific cells

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
X-inactivation transcription activator proteins and this enables specific gene activation in specific cells

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.


X inactivation1
X-inactivation transcription activator proteins and this enables specific gene activation in specific cells

zygote

Inactivation

Embryo

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

This is a Developmental rule that overlays on top of Mendellian rules!


Barr bodies
Barr bodies transcription activator proteins and this enables specific gene activation in specific cells

· 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.

XXY male

XY male

XX female

XXX female


Mosaic expression

X transcription activator proteins and this enables specific gene activation in specific cellsmXf

Xm

Xm

XmXf

Xf

Xf

Xm

Xm

Xf

Xf

Mosaic expression

XmXf

XmXf

XmXf

XmXf

XmXf

XmXf

XmXf

XmXf

XmXf


Tortoise shell cats
Tortoise shell cats transcription activator proteins and this enables specific gene activation in specific cells

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.


Tortoiseshell cats

All tortoiseshell cats are female transcription activator proteins and this enables specific gene activation in specific cells

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

Tortoiseshell cats


Tortoise shell cats1
Tortoise shell cats transcription activator proteins and this enables specific gene activation in specific cells

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


Tortoise shell cats2
Tortoise shell cats transcription activator proteins and this enables specific gene activation in specific cells

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.


Dna methylation is not perfectly inherited during development aging
DNA Methylation is not perfectly inherited during development/aging

Fraga et al., 2005

PNAS 102(30):10604-9.


Xxxxxxxxx
xxxxxxxxx development/aging


Epigenetic inheritance and meiosis
Epigenetic inheritance and meiosis development/aging

sperm

egg

Embryo

Active gene from father maintains activity

Inactive gene from mother remain silent


Imprinting

  • examples: development/aging

    • Developmentally regulated / tissue specific gene expression

    • X chromosome dosage compensation

    • Gene Imprinting

    • Position effect variegation (PEV)

Imprinting

Occurs on Autosomes

Occurs only on some genes on autosomes


Big bottom male x normal true breeding female development/aging

203 big bottom:209 normal

C

N

N

N

C : N

N N

50%

50%

Normal male x 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

Big bottom is autosomal dominant?


CC x development/agingNN

100% Callipyge

NN x CC

0% Callipyge

Calliphyge gene is expressed when inherited from the males!!!

The calliphyge locus from mother is always silenced.


Callipyge

* development/aging

Callipyge

Normal female XNormal male

The callipyge locus from mother is always silenced.

Normal phenotype

female allele is imprinted (turned off) and male allele is expressed

Normal female Xmutant male

mutant female XNormal male

*

Mutant phenotype

Normal allele (from mom) is imprinted (turned off) and mutant allele (from dad) is expressed

Normal phenotype

Mutant allele (from mom) is imprinted (turned off) and normal allele (from dad) is expressed


Imprinting1
Imprinting development/aging

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

Somatic cell

A=off

The original imprint is erased in gametes and the new imprint is established in progeny during gamete formation


Imprinted loci
Imprinted loci development/aging


Dna methylation and imprinting
DNA Methylation and imprinting development/aging

CH3 CH3

Father

IGF2 Gene

Enhancer

CTCF

CTCF

Mother

IGF2 Gene

Enhancer


War of the sexes
War of the sexes development/aging

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.


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


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