Moving to the nucleus ..... Biased transmission of alleles or entire chromosomes Segregation distortion (meiotic drive, selfish DNA) Gametophytic effects in plants Biased or unusual patterns of gene expression Maternal effect genes Imprinted genes (parent-of-origin expression bias) Paramutation (allelic cross-talk & silencing) Epigenetics – Heritable changes in gene expression that do not involve changes in gene sequence • Mediated by changes in chromatin structure (Grelon & Elgin, Nature 447:399) Epigenetics – Mediated by changes in chromatin environment e.g. Position effect variegation (PEV) in Drosophila genes or transgenes located near heterochromatic regions undergo mitotically stable changes in expression due to changes in chromatin organization • An inversion placed the white gene close to the pericentric heterochromatin • Sectors of drosophila white gene off (white eye color) result from heterochromatic regions expanding to include the white gene How might this phenotype be used to investigate the mechanisms of PEV and related gene expression phenomena? (Grelon & Elgin, Nature 447:399) Epigenetics - Factors implicated in heterochromatin formation Repetitive DNA DNA methyln H3K9 methyln HP1 Small RNAs RDR Sp Nc Dm Mm At Yes No Yes Yes Yes Yes Yes Yes Yes Yes No* No* Yes No* Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes No* Yes Yes Yes - factor implicated in heterochromatin formation No - factor is not present in the organism No* - organism has the factor but no role in heterochromatin formation Sp, Schizosaccharomyces pombe; Nc, Neurospora crassa; Dm, Drosophila melanogaster; Mm, Mus musculus ; At, Arabidopsis thaliana RDR, RNA-dependent RNA polymerase; HP1, Heterochromatin Protein 1; Pol II, RNA polymerase II; H3K9, Histone 3- lysine 9 (Grelon & Elgin, Nature 447:399) Epigenetics - RNA silencing mechanisms (RNAi) • Transcriptional (chromatin changes) • Post transcriptional Transcript degradation Translation repression • Share common/core mechanistic features A Nobel-prize winning discovery made in C. elegans! a) Silencing of a gfp transgene via doublestranded gfp RNA b) Mutant defective in RNAi [Fire et al. Nature 391: 806] [Mello & Conte, Nature 431:338] Epigenetics – RNAi mechanisms • Post transcriptional Transcript degradation Translation repression • Share common/core mechanistic features • Note there are lots of routes to dsRNA Nucleus cytosol [Buchon & Vaury Heredity 96:195] Epigenetics – RNAi • Transcriptional (chromatin changes) Nucleus [Buchon & Vaury Heredity 96:195] Epigenetics - RNAi • Transcriptional (chromatin changes) • Post transcriptional Transcript degradation Translation repression • Share common/core mechanistic features • Present in a wide array of organisms How are RNAi pathways adaptive or advantageous? (To the organisms not the molecular biologists!) Epigenetics – Genomic imprinting Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression • Seen in placental mammals and in plants • For some imprinted genes, the maternal allele is silenced & paternal allele expressed • For others, the paternal allele is silenced & maternal allele expressed Epigenetics – Genomic imprinting Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression • Affects the expression but not transmission of alleles • Two gene copies present • One gene copy active = functional haploidy Epigenetics – Genomic imprinting Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression A limited number of genes behave in this way • ~80 of 30,000 human genes • Primarily expressed in embryo and placenta • Plant imprinted genes are expressed in the endosperm • Almost no plant embryo expressed imprinted genes Epigenetics – Genomic imprinting Early evidence from isoparental embryos in mice • created by manipulation of nuclei post fertilization • always lethal • gynogenotes (2 egg-derived genomes) have under-developed placenta • androgenotes (2 sperm-derived genomes) have over-developed placenta What does this say about the contributions of each parental genome with respect to development? Genomic imprinting - Human Igf2-H19 a Insulin growth factor 2 (Igf2) paternally expressed H19 (a non-coding RNA) maternally expressed Regulated by a differentially methylated region (DMR, or imprinting control region ICR) [MacDonald Genetics Res Intl doi:10.1155/2012/585024] Genomic imprinting - Human Igf2-H19 Paternal DMRs methylated - H19 off, Igf2 on • DMR1 is a silencer inactivated by methylation • DMR2 is an enhancer that is activated by methylation Maternal DMRs hypo-methylated - H19 on, Igf2 off • CTCF binds DMR • Downstream enhancer engaged for H19 expression • DMR1 silencer of Igf2 active • DMR2 enhancer of Igf2 is inactive [MacDonald Genetics Res Intl doi:10.1155/2012/585024] Genomic imprinting - Human Igf2-H19 Practice question In mice, complete loss of Igf2 function is viable, the mutant mice are just much smaller than wild-type mice. If a mouse is heterozygous for a loss-of-function mutation at the Igf2 locus (genotype Igf2 – / + ), will this mouse have a mutant or wild-type phenotype? Explain your answer. If an Igf2 – / + male mouse is mated with a wild-type (Igf2 +/+) female mouse, what are the expected frequencies of Igf2 genotypes and resulting phenotypes in the offspring? Explain your answer.. Genomic imprinting in mammals Parent-of-origin gene expression imprints must be correctly set in the gametes every generation! Female embryo Male embryo sperm imprints egg imprints meiosis meiosis • Maternal & paternal imprints are erased in primordial germ cells (PGCs) • Left, reset for a paternal pattern in sperm • Right, reset for maternal pattern in eggs • Failure to re-set correctly leads to developmental disorders in next generation [Kinoshita et al. Sem Cell Devel Biol 19:574] Failure to correctly re-set imprints leads to developmental disorders in next generation e.g. Prader – Willi Syndrome (PWS) / Angleman’s Syndrome (AS) locus • ~ 6 mb deletion human 15q11– q13 • PWS patients (retardation, obesity) inherit 15q11– q13 deletion from paternal parent (functional maternal copy) • AS patients (retardation, seizures) inherit deletion from maternal parent (functional paternal copy) • Some patients have two normal chromosomes but both exhibit maternal expression pattern or paternal expression pattern (imprinting re-set defect) [ Horsthemke and Wagstaff, Am J Human Genet 146A:2041] Genomic imprinting - Arabidopsis endosperm (but not embryo) PHE1 transcription factor • master regulator of seed development • paternally expressed FWA, FIS2, MEA repressive chromatin re-modeling proteins • maternally expressed Differential methylation established in gametophytes • Paternal PHE1 on via 3’ methylation • Paternal FWA, FIS2, MEA off via 5’ methylation • Maternal FWA, FIS2, MEA on - demethylated by DME • Maternal PHE1 off - repressed by MEA-FIS2 chromatin remodeling complex [Kinoshita et al. Sem Cell Devel Biol 19:574] Epigenetics – Genomic imprinting Parent-of-origin in gene expression Plants and animals: • • Genes involved in growth and development Differential methylation (paternal vs maternal) Plants: • • • Occurs in endosperm not embryo Endosperm only (genetic dead-end) does not require “re-setting” each generation Genes are not clustered Animals: • • Occurs in placenta and embryo Must be set for correct sex every generation Complex loci clustering of genes around Cis-acting imprinting control (IC) regions Clusters include paternally and maternally expressed genes Often anti-sense transcription of silenced genes Epigenetics – Paramutation A change in expression of one allele brought about by association with another allele How does this violate Mendel’s law of independent segregation? A paramutagenic allele is able to direct change A paramutable allele is susceptible to change (paramutation) After paramutation, the altered allele also becomes paramutagenic Paramutation is stable through at least one (and sometimes many) subsequent generations A neutral allele is neither paramutable nor paramutagenic Most alleles are neutral! Epigenetics – Paramutation at the B locus of maize B’- paramutagenic allele conditions pale pigment B-I paramutable allele conditions intense purple pigment F1 progeny are all pale! How does this progeny differ from Mendelian expectation? BC1 progeny are all pale! (Chandler and Stam, Nature Rev Genet 5:536) Epigenetics – Paramutation at the B locus in maize Forward genetics provides answers! Screen for mutants that maintain purple pigment in the presence of B’ modifier of paramutation mop1-1 mutation (A) B' Mop1+/mop1-1 (B) B' mop1-1/mop1-1 should look like A; instead looks like C (C) B-I Mop1+/Mop1+ Positional cloning of the affected gene - mop1 encodes an RNA dependent RNA polymerase What mechanism of paramutation is suggested by this finding? (Dorweiler et al. Plant Cell 12:2101; Alleman et al. Nature 42:295) Epigenetics – Paramutation at the B locus of maize • B-I and B’ are identical in sequence • B-I and B’ have 7 tandem repeated copies of an 853 bp sequence motif that is single-copy in neutral alleles • This motif is 100kbp upstream from the B promoter • B-I has open chromatin conformation and B’ has closed chromatin conformation in the repeat region • Small RNAs transcribed from this repeat mediate change in B-I chromatin conformation from open to closed • Important note: In paramutation, the direction of the cross makes no difference! [Arteaga-Vazquez & Chandler Curr Opin Genet Dev 20:156] Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus Kit site-specific knock-out mutation by lacZ insertion (Kit tm1Alf/tm1Alf ) is homozygous-lethal Heterozygote Kit tm1Alf/+ has white tail and feet [Rassoulzadegan et al. Nature 441:469] Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus Kit +/+ x Kit tm1Alf/+ • All progeny white-tailed! • Southern blot confirms recovery of homozygous (Kit +/+) and heterozygous (Kit tm1Alf/+) progeny • Paramutation has altered expression of the Kit+ allele, now designated Kit* +/+ Kit +/+ x Kit tm1Alf/+ [Rassoulzadegan et al. Nature 441:469] Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus Kit tm1Alf/+ and Kit *+/+ : • Reduced polyA Kit transcript relative to Kit+/+ • Increased aberrant transcripts relative to Kit+/+, including small RNAs [Rassoulzadegan et al. Nature 441:469] Non-Mendelian Genetics Unusual patterns of gene transmission • Organelle Genetics • Segregation distortion meiotic drive or “selfish genes” Unusual patterns of gene expression • Maternal effect genes • Epigenetics Small RNAs & gene silencing Imprinting Paramutation