Epigenetic Regulation of Fur Color and Gene Expression in Mice and Cats

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

2. 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 Ectopic Agouti expression causes yellow fur, obesity, diabetes and tumorigenesis Avy can be used as an epigenetic biosensor to study the nutritional and environmental influences on the fetal epigenome from Jirtle and Skinner, Nature Rev.Genet. 8, 253 (2007)

3. The Effect of Nutrition on the Epigenome 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 from Jirtle and Skinner, Nature Rev.Genet. 8, 253 (2007)

4. Progression of Epigenetic Changes in IUGR Rats Pdx1 is a transcription factor necessary for b-cell function 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 from Pinney and Simmons, Trends Endocrinol.Metab. 21, 223 (2009)

5. Imprinting Expression of only one allele of a locus Only ~100 genes in mammals are imprinted Parthenogenesis is not possible in mammals due to incorrect expression of imprinted genes 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 imprinted gene clusters are regulated by methylation-regulated insulators Some clusters of imprinted genes contain long ncRNAs that control allele-specific expression

6. 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 from Bartolomei, Genes Dev. 23, 2124 (2009) In males, the downstream enhancer activates Igf2 and H19 expression is repressed by DNA methylation

7. A long ncRNA Controls Imprinting at the Igf2r Locus 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 from Bartolomei, Genes Dev. 23, 2124 (2009)

8. Imprinting of the PWS-AS Locus from Ferguson-Smith and Surani, Science293, 1086 (2001) 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

9. Dosage Compensation Mechanisms Genomes compensate for different numbers of sex chromosomes by adjusting gene expression levels from Straub and Becker, Nature Rev.Genet.8, 47 (2007)

10. 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 Monoallelic expression of Xist is maintained Xi is reprogrammed in the female germ line from Augui et al., Nature Rev.Genet. 12, 429 (2011) Xi reprogramming correlates with expression of pleuripotency factors

11. 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 Tsix is an RNA expressed from the opposite strand from Xist that acts as an Xist repressor RNF12 activates Xist in a dose- dependent manner by ubiquitylating the REX1 transcription factor from Augui et al., Nature Rev.Genet. 12, 429 (2011) REX1 activates Tsix and represses Xist

12. The Basic Events of X-inactivation from Lee, Genes Dev. 23, 1831 (2009)

13. The Mechanism of Pairing to Initiate 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)

14. Stepwise Progression of X Inactivation in Differentiating ES Cells One X chromosome is converted to facultative heterochromatin Xist transcription off the inactive X initiates chromatin modification events X inactivation is maintained epigenetically from Brockdorff, Trends Genet. 18, 352 (2002)

15. Calico Cats One of the genes controlling fur color is on the X chromosome B – orange b - black Female mammals are genetic mosaics Random X inactivation early in embryonic development leads to patchworks of skin cells expressing each allele

16. The Dosage Compensation Complex in Drosophila 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 from Gilfillan et al., FEBS Lett. 567, 8 (2004) H4K16 acetylation impedes formation of condensed chromatin structure

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

18. 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

19. The 11th Commandment

20. The Replicon Model Sequence elements determine where initiation initiates by interacting with trans-acting regulatory factors from Aladjem, Nature Rev.Genet.5, 588 (2007)

21. Mechanics of DNA Replication in E. coli Leading strand is synthesized continuously and lagging strand is synthesized as Okazaki fragments 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

22. Initiation of DNA Replication in E. coli DnaA binds to high affinity sites in oriB DnaA facilitates the melting of DNA-unwinding element DnaC loads DnaB helicase to single stranded regions DnaB helicase unwinds the DNA away from the origin from Mott and Berger, Nature Rev.Microbiol.5, 343 (2007)

23. DnaB is an ATP-dependent Helicase DnaB unwinds DNA in the 5’-3’ direction DnaB uses ATP hydrolysis to separate the strands SSB proteins prevent the separated strands from reannealing from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-8

24. RNA Primer Synthesis Does Not Require a 3’-OH Primase is recruited to ssDNA by a DnaB hexamer from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-12

25. Coordination of Leading and Lagging Strand Synthesis Two molecules of Pol III are bound at each growing fork and are held together by t 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

26. 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, Nature456, 762 (2008)

27. Replisome Bypass of a Co-directional RNA Polymerase from Pomerantz and O’Donnell, Nature456, 762 (2008)

28. Replisome Bypass of a Co-directional RNA Polymerase Replication fork recruits the 3’- terminus of the mRNA to continue leading-strand synthesis The leading strand is synthesized in a discontinuous fashion from Pomerantz and O’Donnell, Nature456, 762 (2008)

29. Bidirectional Replication of SV40 DNA from a Single Origin from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-32

30. Replication of SV40 DNA T antigen binds to origin and melts duplex and RPA binds to ss DNA Primase synthesizes RNA primer and Pol a extends the primer PCNA-Rfc-Pol d extend the primer from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-31