from Dolinoy, Nutr.Rev. 22 (Suppl. 1), S7 (2008)

The Viable Yellow Agouti Locus

Agouti promotes yellow pigment formation on black hair shaft

Wild-type mice have brown fur due to Agouti expression from hair cell-specific promoter

Avy contains an IAP insertion that contains a promoter expressed in all cells

from Jirtle and Skinner, Nature Rev.Genet. 8, 253 (2007)

Avy is a Metastable Epiallele

Avy can be modified in a variable and reversible manner

Methylation status of IAP determines the activity of the ectopic promoter

Avy can be used as an epigenetic biosensor to study the nutritional and environmental influences on the fetal epigenome

Ectopic Agouti expression causes yellow fur, obesity, diabetes and tumorigenesis

The Effect of Nutrition on the Epigenome

from Jirtle and Skinner, Nature Rev.Genet. 8, 253 (2007)

Feeding of pregnant Avy/a mice with methyl-rich supplements repress the ectopic Avy promoter

Offspring of diet-supplemented mice have brown coat color and methylated IAP

Progression of Epigenetic Changes in IUGR Rats

Pdx1 is a transcription factor necessary for -cell function

from Pinney and Simmons, Trends Endocrinol.Metab. 21, 223 (2009)

Intrauterine growth restriction recruits histone deacetylases that prevents USF-1 binding

Altered histone methylation reinforces Pdx1 repression

Recruitment of DNMT3A locks Pdx1 in a silent state

The result is defective glucose homeostasis

Imprinting

Expression of only one allele of a locus

Only ~100 genes in mammals are imprinted

Most imprinted genes are involved in growth control or postnatal behavior

Imprinted genes involves allele specific methylation and are resistant to genome-wide demethylation in germ cell development

Some clusters of imprinted genes contain long ncRNAs that control allele-specific expression

Some imprinted gene clusters are regulated by methylation-regulated insulators

Parthenogenesis is not possible in mammals due to incorrect expression of imprinted genes

Imprinted Expression of the H19 and Igf2 Genes

ICR is methylated in the male germ line

ICR is protected from methylation in the female germ line by CTCF

CTCF binds to the unmethylated ICR in females and forms an insulator that prevents the activation of Igf2 by a downstream enhancer

In males, the downstream enhancer activates Igf2 and H19 expression is repressed by DNA methylation

from Bartolomei, Genes Dev. 23, 2124 (2009)

from Bartolomei, Genes Dev. 23, 2124 (2009)

ICR in the Airn promoter is methylated in females

Airn is expressed in males and silences Igf2r, Slc22a2 and Slc22a3 in females

Airn is a long ncRNA that might associate with proteins that modify histones

A long ncRNA Controls Imprinting at the Igf2r Locus

from Ferguson-Smith and Surani, Science 293, 1086 (2001)

Imprinting of the PWS-AS Locus

The AS-ICR is required for methylation and inactivation of the PWS-ICR in females to repress nearby genes

The AS-ICR is nonfunctional in males allowing the PWS-ICR to activate nearby genes

The PWS-ICR promotes expression of an antisense Ube3a transcript in males

from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)

Dosage Compensation Mechanisms

Genomes compensate for different numbers of sex chromosomes by adjusting gene expression levels

from Augui et al., Nature Rev.Genet. 12, 429 (2011)

X Chromosome Inactivation in Female Mouse Embryos

Xp is initiatially inactivated after fertilization due to a maternal imprint

A maternal pool of RNF12 initiates imprinted Xp inactivation

Xp is reprogrammed at the blastocyst stage

Random X chromosome inactivation takes place in the ICM due to reactivation of RNF12 from Xp

Xi reprogramming correlates with expression of pleuripotency factors

Monoallelic expression of Xist is maintained

Xi is reprogrammed in the female germ line

The X-Inactivation Center in Mouse

The XIC is the minimum region necessary to trigger X-chromosome inactivation

Xist is an RNA expressed from Xi that coats the X chromosome in cis

RNF12 activates Xist in a dose-dependent manner by ubiquitylating the REX1 transcription factor

REX1 activates Tsix and represses Xist

Tsix is an RNA expressed from the opposite strand from Xist that acts as an Xist repressor

from Augui et al., Nature Rev.Genet. 12, 429 (2011)

from Lee, Genes Dev. 23, 1831 (2009)

The Basic Events of X-inactivation

The two X chromosomes are brought together by CTCF, Tsix and Xite

Transcription factors stochastically shift to the future Xa

Tsix becomes monoallelically expressed

Differential chromatin modifications in Xist lead to its monoallelic expression

from Lee, Genes Dev. 23, 1831 (2009)

The Mechanism of Pairing to Initiate X-inactivation

Stepwise Progression of X Inactivation in Differentiating ES Cells

from Brockdorff, Trends Genet. 18, 352 (2002)

One X chromosome is converted to facultative heterochromatin

Xist transcription off the inactive X initiates chromatin modification events

X inactivation is maintained epigenetically

Calico Cats

One of the genes controlling fur color is on the X chromosome

B – orangeb - black

Random X inactivation early in embryonic development leads to patchworks of skin cells expressing each allele

Female mammals are genetic mosaics

The Dosage Compensation Complex in Drosophila

from Gilfillan et al., FEBS Lett. 567, 8 (2004)

SXL in females prevents MSL2 translation

MSL2 in males stabilizes roX, MSL1, and MSL3

DCC binds to high affinity sites on X chromosome

DCC spreads to nearby sites on active chromatin

H4K16 acetylation impedes formation of condensed chromatin structure

from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)

DCC is Localized to the X Chromosome

DCC localization is determined by staining of polytene chromosomes with anti-MSL1

DCC associates almost exclusively with transcribed regions

DNA Replicates by a Semiconservative Mechanism

Grow cells in 15N and transfer to 14N

Analyze DNA by equilibrium density gradient centrifugation

Presence of H-L DNA is indicative of semiconservative DNA replication

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-29

The 11th Commandment

The Replicon Model

from Aladjem, Nature Rev.Genet. 5, 588 (2007)

Sequence elements determine where initiation initiates by interacting with trans-acting regulatory factors

Leading strand is synthesized continuously and lagging strand is synthesized as Okazaki fragments

Mechanics of DNA Replication in E. coli

The 5’ to 3’ exonuclease activity of Pol I removes the RNA primer and fills in the gap

DNA ligase joins adjacent completed fragments

from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-9

Initiation of DNA Replication in E. coli

DnaA binds to high affinity sites in oriB

DnaC loads DnaB helicase to single stranded regions

DnaB helicase unwinds the DNA away from the origin

DnaA facilitates the melting of DNA-unwinding element

from Mott and Berger, Nature Rev.Microbiol. 5, 343 (2007)

DnaB is an ATP-dependent Helicase

SSB proteins prevent the separated strands from reannealing

DnaB uses ATP hydrolysis to separate the strands

DnaB unwinds DNA in the 5’-3’ direction

from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-8

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-12

RNA Primer Synthesis Does Not Require a 3’-OH

Primase is recruited to ssDNA by a DnaB hexamer

Coordination of Leading and Lagging Strand Synthesis

Two molecules of Pol III are bound at each growing fork and are held together by

The size of the DNA loop increases as lagging strand is synthesized

Lagging strand polymerase is displaced when Okazaki fragment is completed and rebinds to synthesize the next Okazaki fragment

from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-11

from Pomerantz and O’Donnell, Nature 456, 762 (2008)

Interruption of Leading Strand Synthesis by RNA Polymerase

Most transcription units in bacteria are encoded by the leading strand

Natural selection for co-directional collisions in the cell

from Pomerantz and O’Donnell, Nature 456, 762 (2008)

Replisome Bypass of a Co-directional RNA Polymerase

from Pomerantz and O’Donnell, Nature 456, 762 (2008)

Replication fork recruits the 3’-terminus of the mRNA to continue leading-strand synthesis

The leading strand is synthesized in a discontinuous fashion

Replisome Bypass of a Co-directional RNA Polymerase

Bidirectional Replication of SV40 DNA from a Single Origin

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-32

Replication of SV40 DNA

T antigen binds to origin and melts duplex and RPA binds to ss DNA

Primase synthesizes RNA primer and Pol extends the primer

PCNA-Rfc-Pol extend the primer

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-31