1. Genome size and Gene Number
1000Kb=1Mb
1000Mb=1Gb
Number of protein coding genes
Organism Gene number Genome Size Cell number
S. cerevisiae ~6000 (1x) 12Mb (1x) 1
S. pombe ~ Mb
D. melanogaster ~14,000 (2x) 175Mb (14x)
C. elegans ~19, Mb 1000
M. musculus ~22, Gb 1013
H. sapiens ~22,000 (4x) 3.3Gb (275x) 1014 (+ 50x bacteria)
Number of genes does not scale across species despite increases in developmental and cognitive complexity. Amount of repetitive DNA increases.
Genome size increases by duplication and transposition. It is the raw material for evolution.
Transposition mediates dissemination of functional cassettes and re-structuring of regulatory networks enabling phenotypic divergence

2. Complexity and Regulation
Lack of gene scaling
Gene number does not increase dramatically
Limited proteomic diversification through evolution
Gene size does not increase
Amount of DNA between genes increase
Increase in Regulatory complexity
Increase in the number and complexity of regulatory sequences,
Increased in regulatory molecules
Alterations in expression patterns of regulatory molecules
Increased inter-connected networks and Combinatorial control by transcription factors

3. Epigenetic Inheritance: DNA sequence is same
Classical Mendelian Inheritance of Phenotypic traits: results from allelic differences caused by mutations in DNA sequence
Epigenetics
Epigenetic Inheritance: DNA sequence is same
Epigenetics1:
Differential expression of both alleles in different cells due to stable regulation during development YY Yy yY yy
There are two copies of a gene in diploid organisms
Both allelic copies are expressed in cells YY Yy yY yy
Yellow pea seed gene is only expressed in seeds, not roots etc
Epigenetics2:
DNA sequence is same.
Expression of only one allele of two alleles within a cell in entire organism YY Yy yY yy
The study of phenomenon and mechanisms that cause “heritable” changes to gene expression that are not dependent on changes in DNA sequence and that are mediated by chromosome-bound soluble factors

4. Modern Definition
Nanney in 1958 used the term epigenetic to indicate that soluble components were responsible for maintaining and perpetuating expression states of genes through replication and cell division.
Holliday in 1994 provided a definition for Epigenetics-
1 “study of changes in gene expression which occur in...differentiated cells and the mitotic inheritance of the given patterns of gene expression”
2 “Nuclear inheritance which is not based on differences in DNA sequence”
“Study of changes in gene expression that are mitotically and/or meiotically heritable and that do not entail change in DNA sequence”
The study of phenomenon and mechanisms that cause chromosome-bound heritable changes to gene expression that are not dependent on changes in DNA sequence

5. Variation in Appearance
Mutation/Alteration
Packaging/Regulation
Genetic inheritance refers to the transmission of DNA based information from one generation to the next.
Epigenetic information is superimposed on DNA sequence information and modifies the readout of the data
Dual inheritance systems in organisms, one of which is genetic and the other epigenetic.
Stable Phenotypic difference between two cells due to change in DNA sequence between the two cells
Semi-Stable Phenotypic difference between two cell types in the absence to any changes in the DNA sequence of those cell types
Molecular mechanism:
Decorations on the DNA establish and stably propagate patterns of gene expression

6. Epigenetics: Gene regulation through stable activation/repression
Heritable changes in gene function that cannot be explained by changes in gene sequences
During early development there is a progressive restriction of cellular plasticity accompanied by acquisition of cell type specific patterns of gene expression and modifications on genes
Epigenetic factors impose restrictions to the plasticity of totipotent embryonic cells
Epigenetic factors impose a cellular memory that accompanies and enables stable differentiation

7. Epigenetic inheritance during mitosis
egg
sperm
Embryo
All Genes are poised for activity
Cell commitment
Specific genes activated
All other genes inactivated
Ecto
Meso/Endo
Specific genes maintain activity
Other genes remain silent
Specific genes maintain activity
Other genes remain silent
Mechanism:
Presence of Transcription factors

8. Cell specific Activation
Different Enhancers bind different tissue and cell specific transcription activator proteins and this enables specific gene activation in specific cells
HNF3
Hepatocyte
Liver gene1
Liver gene2
Brain gene1
Brain gene2
Neurons
NZF2
Liver gene1
Liver gene2
Brain gene1
Brain gene2

9. Transcription activator +++ ---
One set of genes active in one cell type and another set of genes active in a different cell type
How is selective regulation of different sets of genes achieved in different cell types-
Specific Transcription factors.
Once established this state is maintained through repeated cell growth and cell divisions
Cell, Volume 152, Issue 6, 2013,
Active Inactive
Transcription activator

10. Function of Transcription factors
Enhancers activate transcription of genes
Cell/tissue specific transcriptional activators bind to enhancers of genes that have binding sites for these factors
-Help recruit enzymes that modify chromatin at the promoter
- Recruit general transcription factors and RNA polymerase
Inr
TATA
Gene
Promoter
Enhancer

11. Specific genes activated All other genes inactivated
sperm
egg
Embryo
All Genes are poised for activity
Cell commitment
Specific genes activated
All other genes inactivated
Ecto
Meso/Endo
Active genes maintain activity
Inactive genes remain silent
Active genes maintain activity
Inactive genes remain silent
Mechanism of repression:
Absence of Transcription factors
Presence of Repressors
Changes in Chromatin structure
Changes in DNA methylation

12. 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
Liver gene2
Brain gene1
Brain gene2
Brain Cell
NZF2
Liver gene1
Liver gene2
Brain gene1
Brain gene2

13. Transcription activator +++ --- Repressor proteins --- +++
Gene Repression
Heritable changes in gene expression that do not involve changes in DNA sequences
All Genes not active in all cells
Active Inactive
Transcription activator
Repressor proteins
Specific Histone Modi
Specific Histone Modi
DNA methylation

14. Mechanism of gene repression
Prm
ORC
Rap1
Abf1 1 2 4 3
Silencer 3 1 2 4
ORC
Rap1
Abf1 Ac
Insulator

15. Transcriptionally Active Inactive chromatin
Euchromatin
Active
Heterochromatin
Inactive
Euchromatin
Less condensed, rich in genes
Transcriptionally active
Constitutive heterochromatin: Repetitive DNA (Satellite) -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
Facultative heterochromatin:
The rest is transcriptionally inactive
Can be activated in certain tissues or developmental stages
These inactive regions are known as “facultative heterochromatin”

16. Amount of heterochromatin in different cells
Chromosomes carry developmental program- composed of DNA + proteins
Active genes are bound by specific proteins.
Silent genes are bound with different specific proteins
Gene that are active reside in the interior of the nucleus
Gene that are silent reside at nuclear periphery
Combination of these two phenomenon affects expression of genes
Rat Gut epithelia
Rat Liver
Rat lymphocyte
Rat egg

17. There was no loss or gain of genetic material
PEV in Flies
Muller (1930) described Drosophila mutations that involved chromosomal translocations-
There was no loss or gain of genetic material
Flies had mottled variegated eyes.
Chromosome regions and not individual genes affected this trait
Genes were not independent entities. Gene function affected by chromosomal location.
White
Inversion

18. What is Partial Silencing
Completely Repressed
Completely Derepressed
Partially Derepressed

19. X chromosome Inactivation
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
2x 1x
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 chromosomes and causes chromosome condensation which reduces transcription.
In mammals, X chromosome inactivation occurs in females by formation of heterochromatin on one X chromosome

20. Mammalian X-chromosome inactivation
Mammalian males and females have one and two X chromosomes respectively.
XY XX
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.

21. Dosage compensation
Mammalian males and females have one and two X chromosomes respectively.
XY XX
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 (facultative) and forms a structure called a Barr-body.
XCI is random. It occurs at the 500 cell stage of the embryo (during development). For a given cell in a developing organism, the 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.

22. 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
XXY male
XY male

23. Inactivation and inheritance in all cells in the bodyXY XX
reactivate X
egg X
sperm XX XX
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

24. 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 silenced
Tsix Active
Xist Active
Tsix RNA
Xist RNA
Xist RNA
Tsix RNA
Pair
Coat inactive X
- methylate DNA

25. Marking DNA- How??? Protein Binding to DNA DNA Modification A C G T A

26. Gene on X-chromosome and Mosaic expression
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf
XmXf Xm Xf

27. Tortoise shell cats
Orange
Black
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.

28. 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
OO x oY
F1 females are Oo

29. 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.

30. xxxxxxxxx

31. Epigenetic inheritance and Parents
egg
sperm
Embryo
Active gene from father maintains activity
Inactive gene from mother remain silent

32. Occurs only on some genes on autosomes
examples:
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

33. Big bottom male x normal true breeding female
203 big bottom:209 normal C N
C : N
N N
50% N C
Normal male x big bottom female
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?

34. CC x NN 100% Callipyge NN x CC 0% Callipyge
Calliphyge (mutant) gene is expressed in offspring when inherited from the males!!!
The calliphyge (mutant) gene from mother is always silenced in offspring

35. The callipyge gene from mother is always silenced.
Normal female X Normal male
Normal phenotype
female allele is imprinted (turned off) and male allele is expressed
The callipyge gene from mother is always silenced.
Normal female X mutant male
mutant female X Normal male *
Normal phenotype
Mutant allele (from mom) is imprinted (turned off) and normal allele (from dad) is expressed *
Mutant phenotype
Normal allele (from mom) is imprinted (turned off) and mutant allele (from dad) is expressed

36. A small number of genes (~200) on autosomes
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
Somatic cell
a=off
The original imprint is erased during gamete formation and the new imprint is established in progeny

37. Imprinted loci

38. DNA Methylation and imprinting in offspring
CTCF
Enhancer
IGF2 Gene
Father’s
chromosome
CH3 CH3
Insulator
Boundary model of the IGF-2 and H19 cluster on chromosome 11p15. IGF-2 and H19 share a common enhancer downstream of H19. On the maternal allele, the ICR upstream of H19 is unmethylated and binds the vertebrate enhancer-blocking protein CTCF, which inhibits the activation of the IGF-2 promoter by the enhancer. On the paternal allele, the H19 promoter and ICR are methylated, thus silencing H19 and interfering with CTCF binding. The IGF-2 promoter is activated by its enhancer (30 , 31) .
Enhancer
CTCF
Mother’s
chromosome
IGF2 Gene

39. Why are perfectly good genes turned off?
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.