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

Kinship Theory of Imprinting Conflict exists between the interests of the paternal and maternal genes Paternally-expressed genes generally stimulate growth Maternally-expressed genes generally repress growth For optimal fitness of the father, paternal genes maximize acquisition of maternal resources to ensure larger sized offspring Maternal genes are sparing in the demands of maternal resources, so that the mother has a better chance to bear further offspring

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)

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 Lee, Genes Dev. 23, 1831 (2009) The Onset of X-inactivation

The Basic Events of X Chromosome Inactivation Xist RNA is transcribed from Xi The Xic is the minimum region to trigger X inactivation Xist coats X i in cis Tsix is expressed from the opposite strand from Xist and acts as an Xist repressor Xist binds PRC2 and methylates H3K27 from Lee, Science 338, 1435 (2012)

from Augui et al., Nature Rev.Genet. 12, 429 (2011) X Chromosome Inactivation in Female Mouse Embryos X p is initially inactivated after fertilization due to a maternal imprint A maternal pool of RNF12 initiates imprinted X p inactivation X p is reprogrammed at the blastocyst stage Random X chromosome inactivation takes place in the ICM due to reactivation of RNF12 from X p X i reprogramming correlates with expression of pleuripotency factors Monoallelic expression of Xist is maintained X i is reprogrammed in the female germ line

The two X chromosomes are brought together by CTCF, Tsix and Xite Transcription factors stochastically shift to the future X a 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

Tsix is expressed from one allele and recruits Dnmt3a to silence Xist RepA recruits PRC2 to Xist Tsix blocks recruitment of PRC2 to Xist by RepA Xist-PRC2 spreads in cis Regulation of Xist Expression from Lee, Science 338, 1435 (2012)

Tethering Xist to X i from Lee, Science 338, 1435 (2012)

The early binding sites for Xist are spatially close to the Xist transcription site Xist spreads by exploiting three- dimensional chromosome topology The few genes that escape silencing loop out of the condensed core from Dimond and Fraser, Science 341, 720 (2014) Xist Spreads to Coat the Inactive X Chromosome

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 – orange b - 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 15 N and transfer to 14 N 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, 6 th ed. Fig 4-29

The 11 th 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, 4 th 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, 4 th ed. Fig 12-8

from Alberts et al., Molecular Biology of the Cell, 4 th 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, 4 th 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, 6 th ed. Fig 4-32

A double hexamer of large T antigen binds to SV40 origin Two single hexamers unwind dsDNA and translocates along ssDNA in a 3’-to-5’ direction The helicase can overcome a covalent block to unwinding from Trakselis and Graham, Nature 492, 195 (2012) Mechanism of DNA Unwinding by Large T Antigen

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, 6 th ed. Fig 4-31