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

Kinship Theory of Imprinting Conflict exists between the interests of the paternal and maternal genes 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 Paternally-expressed genes generally stimulate growth Maternally-expressed genes generally repress growth

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

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 Airn is a long ncRNA that might associate with proteins that modify histones from Bartolomei, Genes Dev. 23, 2124 (2009)

Imprinting of the PWS-AS Locus from Ferguson-Smith and Surani, Science 293, 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

Sex Specific Methylation of Imprinting Control Regions Two waves of demethylation and methylation DNA is demethylated during germ cell migration and remethylated in post migration germ cells DNA is demethylated in cleavage stage embryos Differentially methylated regions of ICRs escapes demethylation from Edwards et al., Epigenetics Chromatin 10,:23 DOI 10.1186/s13072-017-0130-8 (2017)

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)

X-inactivation in Mice and Humans from Lee, Genes Dev. 23, 1831 (2009)

X Inactivation and Reactivation During Development Xp is inactivated at the 2-4 cell stage A maternal imprint (H3K9me3 on the Xist promoter) prevents Xm from inactivation Xp reactivation occurs in the preimplantation epiblast and Is correlated with pleuripotency Differentiation triggers random XCI in the epiblast cells of the blastocyst Random XCI is clonally transmitted during mitosis Germ cells are derived from the epiblast from Pasque and Plath, Curr.Opin,Cell Biol. 37, 75 (2015) XCR occurs in primordial germ cells during migration and colonization of the gonads

Model System for Studying Random X Inactivation from Pasque and Plath, Curr.Opin,Cell Biol. 37, 75 (2015) Mouse ESCs derived from the inner cell mass undergo random XCI during differentiation Both X chromosomes are active in ESCs

XCI and XCR are Linked to Differentiation and Pleuripotency from Pasque and Plath, Curr.Opin,Cell Biol. 37, 75 (2015) Expression of pleuripotency-associated transcription factors in fibroblasts results in reprogramming to iPS cells Generation of iPS cells correlates with XCR

X Inactivation is Accompanied by Heterochromatin Formation Xist transcription of the inactive X initiates chromatin modification events Chromatin changes occur in a stepwise manner Chromatin changes are reversed during X chromosome reactivation or somatic cell reprogramming from Brockdorff, Trends Genet. 18, 352 (2002)

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

Regulation of Xist Expression Xist expression is repressed by Tsix transcription Pleuripotency factors repress Xist expression Rnf12 stimulates Xist expression in a dosage-dependent fashion by promoting Rex1 degradation Two active Xs are necessary to trigger XCI by stimulating Rnf12 expression from Galupa and Heard, Curr.Opin.Genet.Dev. 31, 57 (2015) XCI is necessary for differentiation

Identification of Proteins that Bind Xist in vivo UV-crosslink SILAC-labelled ES cells after induction of Xist Purify Xist using a biotinylated antisense probe in denaturing conditions to disrupt non-covalent interactions Separately purify proteins that bind to a control RNA Identify proteins that specifically bind Xist by mass spectrometry and calculation of a SILAC ratio for each protein based on the intensity of all heavy and light peptides originating from Xist or the control RNA from McHugh et al., Nature 521, 232 (2015)

Initiation of X-Inactivation from McHugh et al., Nature 521, 232 (2015) SAF-A binds Xist and tethers Xist to Xi SHARP binds to Xist and recruits SMRT to Xi Xist-SHARP-SMRT recruits or activates HDAC3 Histone deacetylation compacts chromatin and silences transcription PRC2 is recruited to Xi to maintain silencing

Xist Spreads by Proximity Transfer The early binding sites for Xist are spatially close to the Xist transcription site Xist interacts with the lamin B receptor changing the 3D structure of Xi SMCHD1 suppresses TADs by repelling other architectural proteins (CTCF) The few genes that escape silencing loop out of the condensed core from Dimond and Fraser, Science 341, 720 (2014)

Topological Differences Between Xa and Xi Xa has a normal TAD structure Xi consists of two large superdomains whose border binds CTCF Xist repels cohesins which alters the overall structure of Xi 12-20% of human genes on the X chromosome escape inactivation Escapees show local enrichment of CTCF binding sites and are located at the periphery of X i from Finestra and Gribnau, Curr.Opin. Cell Biol. 46, 54 (2017)

Female Mammals are Genetic Mosaics 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 Fur color is an example of cellular memory

The Dosage Compensation Complex in Drosophila SXL in females prevents MSL2 translation MSL2 in males stabilizes roX, MSL1, and MSL3 DMSL complex 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) MOF mediates H4K16 acetylation

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)

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

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

Initiation of DNA Replication in E. coli ATP-DnaA is loaded onto OriC and unwinds the DNA duplex DnaB helicase is loaded, migrates, and expands ssDNA region Primase is loaded and synthesizes RNA primers from Katayama et al., Nature Rev.Microbiol. 8, 163 (2010) Pol III and DnaN clamp is loaded onto the primed sites

Inactivation of Initiation Following Onset of DNA Replication from Katayama et al., Nature Rev.Microbiol. 8, 163 (2010) SeqA binds oriC and inhibits initiation by GATC methylation and transcriptional repression of dnaA Replication results in hydrolysis of ATP-DnaA by Hda datA locus binds ATP-DnaA to compete with oriC DARS1 and DARS2 promote ATP for ADP exchange on DnaA to regulate initiation

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

Coordination of Leading and Lagging Strand Synthesis from Garcia-Muse and Aguilera, Nature Rev.Mol.Cell Biol. 17, 553 (2016) In eukaryotes, the MCM helicase opens the double helix Pole and Pold extend the leading and lagging strands Pola-primase initiates DNA synthesis ssDNA on the lagging strand is coated by RPA Topoisomerases alleviate supercoiling

Replication Fidelity Replicative polymerases incorporate 1 error/107 nucleotides Polymerase can dissociate after incorporating incorrect nucleotide Nascent strand containing error is transferred to exonuclease site Error-containing strand can be extended which can be corrected from Ganai and Johansson, Molecular Cell 62, 745 (2016)

Tautomer Formation Allows Mispairing During DNA Synthesis DNA bases can exist in a disfavored tautomeric form DNA polymerases require an optimal alignment to ensure efficient catalysis The alignment can occur with G-T assumes one of the disfavored tautomeric forms The majority of misincorporation is attributable to tautomer formation from Goodman, Nature 554, 180 (2018)

Consequences of DNA Polymerase-RNA Polymerase Collisions from Garcia-Muse and Aguilera, Nature Rev.Mol.Cell Biol. 17, 553 (2016) DNA replication/transcription conflicts are a source of genomic instability Head-on collisions cause replication fork collapse and dsb RNA polymerase is displaced upon a co-directional collision Topoisomerases prevent collisions by attenuating polymerase progression Genome-wide bias toward co-orientation of DNA replication and transcription in bacteria

Replisome Bypass of a Co-directional RNA Polymerase from Pomerantz and O’Donnell, Nature 456, 762 (2008)

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, Nature 456, 762 (2008)

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

Mechanism of DNA Unwinding by Large T Antigen 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)