EPIGENETIKA MB150P85

1. Přírodovědecká fakulta UK EPIGENETIKA MB150P85 Petr Svoboda mail: svoboda1@natur.cuni.cz tel: 241063147 blatny.com/Epigenetika2007

2. X-INACTIVATION

3. Costs and benefits of sex • automatic two-fold cost of sex over asexual reproduction • “A population of assexually-reproducing females would have twice the growth rate of an otherwise equivalent sexually-reproducing population.” • - sexual reproduction must have some compensating advantage • - sex can accelerate the rate of evolution • - genome maintenance

4. A population of sexually reproducing organisms can, under some conditions, evolve faster than a similar number of asexual organisms. A famous diagram illustrates the argument, which contends that sexual reproduction enables beneficial mutations of different genotypes to be combined into single genotype. This argument was first put forward by Fisher, who concluded that sexual populations have a more rapid rate of evolution than would an otherwise equivalent group of asexual organisms. Fisher's conclusion depends on the rate of mutation: • If favorable mutations are rare, each one will have been fixed in the population before the next one arises. New favorable mutations will always arise in individuals that already carry the previous favorable mutation. Sexual and asexual populations then evolve at the same rate. • If favorable mutations arise more frequently, Fisher's argument works: the sexual population evolves faster. Each new favorable mutation will usually arise in an individual that does not already possess other favorable mutations; the greater speed with which the different favorable mutations combine together causes the sexual population to evolve faster. The higher the rate at which favorable mutations are arising, the greater the evolutionary rate of a sexual relative to an asexual population.This is a case of group selection because it claims that the cost of sex is more than made up for by the faster evolution of the sexual population, or group. Figure: evolution in (a) asexual and (b) sexual populations. The mutations A, B and C are all advantageous. In the asexual population, an AB individual can arise only if the B mutation arises in an individual that already has an A mutation (or vice versa.) In the sexual population, the AB individual can be more easily formed by breeding of a B mutation-bearing individual with an A mutation-bearing individual. (c) If favorable mutations are rare, each will have been fixed before the next mutation arises, and sexual populations will not evolve more rapidly.

5. Costs and benefits deleterious mutations parasitic sequences favourable mutations haploid diploid sexual asexual

6. Evolution of sex chromosomes - many different sex-determining systems in plants and animals with separate sexes. - in some species, sex is determined by environmental factors (control expression of genes leading to male or female development) - other species have evolved genetic systems involving specialized sex chromosomes. - sex chromosomes have arisen independently in many animal groups. Also found rarely in plants. It looks like sex chromosomes were once homologs (a pair of equivalent autosomes—the non-sex chromosomes) that evolved different morphology and gene content because they lost their ability to recombine. Suppression of recombination is thought to start around the sex-determining region, but may eventually affect much of the sex chromosomes. In the absence of recombination, the two chromosomes of a pair evolve separately and one of the often deteriorates. Unequal genetic load must be then compensated by some mechanism.

7. Extreme variability in regulation of sex determination >350 MYA MAMMALIA Mus Xenopus AMPHIBIA >400 MYA CHORDATA Danio PISCES >600 MYA DEUTEROSTOMES Strongylocentrotus ECHINODERMATA COELOMATES PROTOSTOMES ARTHROPODA Drosophila EUMETAZOA Caenorhabditis PSEUDOCOELOMATES NEMATODA

8. XX/XY sex chromosomes - females have two of the same kind of sex chromosome (XX) - males have two distinct sex chromosomes (XY). - found in most mammals and insects (Drosophila). - mammals have a SRY gene on the Y chromosome that determines maleness - fruit fly use the presence of two X chromosomes to determine femaleness. XX/X0 sex determination - females have two copies of the sex chromosome (XX) - males have only one (X0). The 0 denotes the absence of a second sex chromosome. - found in numerous insects (grasshoppers, crickets, and cockroaches) and other invertebrates. - C. elegans: male with one sex chromosome (X0); hermaphrodite with a pair of chromosomes (XX). ZW sex chromosomes - ZW sex-determination system is reversed compared to the XY system - females have two different kinds of chromosomes (ZW) - males have two of the same kind of chromosomes (ZZ). - found in birds and some insects (Lepidoptera) and other organisms. Genes in the ZW region in birds are autosomal in mammals, and vice-versa; therefore, it is theorized that the ZW and XY couples come from different chromosomes of the common ancestor. A paper published in 2004 (Frank Grützner et al, Nature; DOI:10.1038/nature03021) suggests that the two systems may be related. According to the paper, platypuses have a ten-chromosome–based system, where the chromosomes form a multivalent chain in male meiosis, segregating into XXXXX-sperm and YYYYY-sperm, with XY-equivalent chromosomes at one end of this chain and the ZW-equivalent chromosomes at the other end.

11. Different strategies to compensate unequal genetic load upregulation of expression in males XX/XY silencing of one chromosome in females XX/XY reducing expression of both chr. in females XX/X0 Straub 2007

12. Different strategies to compensate unequal genetic load Straub 2007

13. XX/XO Caenorhabditis elegans Stothard 2003 - reducing expression of both chromosomes in females - X:A ratio determines sex and dosage compensation - dosage compensation complex (DCC) - at least poly10 peptides

14. XX/XO Caenorhabditis elegans Stothard 2003 condensin complexes function during mitosis and meiosis for DNA compaction and sister chromatid resolution

15. XX/XY Drosophila melanogaster - upregulation of expression in males - MSL complex, H4K16ac Lucchesi 2005 Amrein 2000 HAT activity MSL - male-specific lethal DCC - dosage compensation complex

16. Homo sapiens/Mus musculus XX/XY - dosage compensation by inactivating one X in female cells 4 steps Counting - if more than one, choose to inactivate, so one remains active Choice - random vs. non-random Initiation - inititation and propagation of chromosome-wide silencing Maintenance - throughout subsequent cell division XCI = X chromosome inactivation Xi = inactive X Xa = active X Xic = X inactivation center Xce = X-controlling element (Xist/Tsix) 2 types of X-inactivation Imprinted X-inactivation - in early embryos, extraembryonic lineage (trophoblast and primitive endoderm) Random X-inactivation - in the epiblast, completed by 5.5.-6.5 dpc

17. Thorvaldsen 2006 X-inactivation and reactivation escape in PGCs imprinted imprinted imprinted random escape

18. X-inactivation during preimplantation development Imprinted X-inactivation • inactive X inherited or de novo silencing after fertilization? • pre-inactivation hypothesis • sex chromosome inactivation during spermatogenesis • XY body in spermatocytes, MSCI (meiotic sex chr. inact.) • MSCI not fully understood, different from XCI (Xist independent, specific chromatin modifications including histone variant H2AX) • staining of 2-cell embryos indicate lack of active transcription on the paternal X • some data support reversion into the active state after meiosis • “de novo” model • Xp active at fertilization, silenced later • staining of 2-cell embryos showing biallelic expression • Xist dependent (Xist expressed at the 2-cell stage)

19. Xist and Tsix

20. Avner 2001

21. Xist and Tsix http://bioweb.wku.edu/courses/biol566/L9XchromSilencing.html Figure 1. Mouse and Human Xic/XIC and Xist/XIST. A. area surrounding the XIST/Xist gene on human and mouse X-chromosomes. Human domain is inverted from mouse relative to telomere. The identification of a human Tsx homolog is unclear. B. Comparison of the mouse Xist and human XIST genes. * = alternative splicing sites. Mouse has a second promoter that has not been found in other Xist/XIST genes analysed to date. Extensive alternative splicing of the human gene has been described yielding isoforms that lack exon 4, half of exon6, exon7 or include the last two introns.

22. http://bioweb.wku.edu/courses/biol566/L9XchromSilencing.html Xist Longest Xist 17.9 kb. Longest XIST 19.3 kb. Mouse and human Xist/XIST show 49% sequence identity which is lower than 5' & 3' UTR regions but slightly higher than introns. Several short stretches of high homology and six repeated elements A-F. No open reading frame. Must operate as polyadenylated RNA.

23. http://bioweb.wku.edu/courses/biol566/L9XchromSilencing.html Xist

24. http://bioweb.wku.edu/courses/biol566/L9XchromSilencing.html Tsix • two promoters and two polyadenylation sites. no significant open reading frames. • Tsix transcripts of up to 4 kb can be produced by splicing. • Tsix is not the counting element (Males lacking Tsix do not inactivate). • Tsix RNA is antisense to Xist and reduces its steady-state level while subsequently promotes Xa choice by increasing the affinity of the cis-linked counting element for blocking factor. • the spliced form of Tsix RNA contains only 2 kb of overlap with the mature Xist transcript. This overlap occurs within a domain of Xist that is critical for silencing activity • antisense transcription throught the Xist sequence is important (Deletion mutants lacking the overlap)

25. Avner 2001

26. Tsix

28. X-inactivation during preimplantation development Imprinted X-inactivation • paternal Xist expression activated at the 2-cell stage • Xist silencing of the maternal X (Xm) is unclear • Tsix (maternal) is detected first at the 8-cell stage • Xist accumulates on the Xp (initiation event) from the 4-cell on • initial chromatin changes found during the 8-32-cell stages • hypoacetylation H3K9 • hypomethylation H3K4 • EED/EZH2 enrichment mediates H3K27 methylation on the Xi • initiation vs. maintenance changes unknown • gradient of silencing from the Xic suggests that silencing is progressive, mediated by Xist RNA spreading • ICM cells reverse imprinted XCI, trophectoderm cells maintain it

29. X-inactivation during preimplantation development Random X-inactivation • paternal Xist silencing reversed after early blastocyst • Xist dispersed or absent • no EED/EZH2 association, • random X-inactivation initiates during implantation and is complete around day 6.5 dpc • initiation is characterized by downregulation of Tsix and upregulation of Xist • Xist coats the Xi in cis • chromatin modifications • DNA methylation is a late step • once established, the Xi is clonally propagated such that females are functionally mosaic for X-linked traits. • epigenetic modification can later maintain Xi repression in a Xist-independent manner

30. Thorvaldsen 2006 Xist and Tsix XCI = X chromosome inactivation Xi = inactive X Xa = active X Xic = X inactivation center Xce = X-controlling element (Xist/Tsix) Avner 2001 doesn’t seem to be the case

31. http://bioweb.wku.edu/courses/biol566/L9XchromSilencing.html XCI in differentiating female ES cells MacroH2A is recruited to the Xi by Xist. MacroH2A followed by exclusion of H2ABBD from Xi. (formation of Barr body). Xist/XIST espression is not necessary to continue Xi after establishment. DNA methylation appears to be extremely important for the stability and maintenance of gene silencing on Xi. DNA methylation concerns promoter regions, overall is the inactive X hypomethylated!

32. Turner syndrome - 45, X or 46, X, abn X - fairly common (10% od spontaneous abortions) - 1 of 40 develops to birth, then the phenotypic effects are relatively mild because each cell has a single functioning X chromosome like those of XX females. - phenotypic female with gonadal dysgenesis and sexual immaturity, have primary amenorrhea (failure to menstruate), infertility, short stature, webbed neck, increased carrying angle at the elbow, cardiovascular and renal abnormalities - 45,X in more than half the patients Number of Barr bodies = zero. Incidence: 1 of 2500 female birhts Why does Turner syndrome occur at all, since only one X chromosome is normally active? There are two active X chromosomes during ovarian development, and certain genes appear to need to be active for normal ovarian function. Turner syndrome oocytes virtually gone by the age of 2 years

33. Klinefelter syndrome - 47, XXY(48, XXXY) - males (Y chromosome). - the phenotypic effects of the extra X chromosomes are mild because, the extra Xs are inactivated and converted into Barr bodies - male with small testes, hyalinized testicular tubules, and azoospermia (failure to produce normal amounts of sperm), resulting in infertility and variable signs of hypogonadism, social pathologies, somewhat reduced IQ, postpubertal testicular failure - may have additional X chromosomes, if so, more likely to be mentally retarded - demonstration in humans that sex is determined by presence or absence of Y chromosome, rather than number of X chromosomes Number of Barr bodies = extra X’s inactivated Incidence: 1 of 1000 male births

34. XYY syndrome - found as 47,XYY, or 48,XXYY 47,XYY - occurs 1/1000 in male live births - occurs 4-20 per 1000 inmates 48,XXYY - 50 times higher in prison inmates than in newborn population - aneuploidy of the Y chromosome must arise from meiotic nondisjunction in the father XX males - incidence 1 in 20,000 - have X-Y interchange - Sry transgenic mice, XX become male XXX, XXXX, XXXXX females - mild phenotypic effects because in each cell all the extra X chromosomes are inactivated. - number of Barr bodies = number of X chromosomes minus one.

35. Turner 2007 Meiotic Sex-Chromosome Inactivation

36. Turner 2007 Meiotic silencing of unsynapsed chomatin

37. IMPRINTING

38. Discovery of Imprinted Genes - experimental manipulation of mouse embryos in the early 1980's showed that normal development requires the contribution of both the maternal and paternal genomes. - gynogenetic embryos (two female genomes) show relatively normal embryonic development, but poor placental development. - androgenetic embryos (two male genomes) show very poor embryonic development but normal placental development. - it is now known that there are around 100 imprinted genes in humans and mice, many of which are involved in embryonic and placental growth and development - the gynogenetic embryos have twice the normal level of maternally expressed genes, and completely lack expression of paternally expressed genes, whereas the reverse is true for androgenetic embryos. - no naturally occurring cases of parthenogenesis exist in mammals - manipulation of a paternal methylation imprint controlling the Igf2 locus allowed the creation of rare individual mice with two maternal sets of chromosomes (not a true parthenogenote).

39. http://atlasgeneticsoncology.org/Deep/GenomImprintID20032.htmlhttp://atlasgeneticsoncology.org/Deep/GenomImprintID20032.html Mouse germ cell pronuclear transplant experiments convincingly demonstrate a different agenda for sperm- versus egg-derived nuclear genomes during development. Development in the absence of a sperm-derived genome (middle column) shows fairly good development of the embryo proper but failed development of the trophoblast lineage. Development in the absence of an egg-derived genome (right column) shows failed development of the embryo proper but exuberant trophoblast growth.

41. Imprinting is a cause of phenotypes in uniparental disomies In 1980 Engel introduced the concept of uniparental disomy (UPD). Uniparental disomy (UPD) arises when an individual inherits two copies of a chromosome pair from one parent and no copy from the other parent. Recall that normally a baby inherits one copy of each chromosome from his/her mother and one copy of each chromosome from his/ her father. In the rare circumstance of UPD a baby may have two copies of one of his/ her mother’s chromosome and no copies of that chromosome from his/ her father. This is called maternal UPD. Paternal UPD is when a child inherits two copies of a specific chromosome from his/ her father and no copies of that chromosome from his/ her mother. This abnormality in inheritance may lead to health concerns in a child. UPD can result in rare recessive disorders, or developmental problems due to the effects of imprinting. UPD may also occur with no apparent impact on the health and development of and individual. We will discuss the effects of UPD in greater detail, but first we must understand how UPD occurs. Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome

42. http://www.mgu.har.mrc.ac.uk/research/imprinting/imprin-viewmaps.htmlhttp://www.mgu.har.mrc.ac.uk/research/imprinting/imprin-viewmaps.html Maps of imprinted genes

46. General features of imprinted genes - typically clustered, clusters may contain monoallelic expression of genes from each parent. - clusters contain imprint control regions and a non-coding RNA is often found associated with it (H19, Air …) - ICRs show parent-of-origin dependent epigenetic modifications (methylation) - many related to growth control (battle of the sexes hypothesis) - it has been reported that imprinted genes tend to have smaller introns. - some genes imprinted only in neural tissues

48. Robertson 2005 Mouse distal 7 imprinted region

49. H19 - a noncoding RNA!! CTCF http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=604167 - CCCTC binding factor with 11 zinc fingers - highly conserved in vertebrates (93% identity human-avian) - binds to regulatory sequences in numerous loci, including H19/IGF2 - binding is methylation sensitive, protects DNA from methylation - insulator - regulates access of enhancers/separates functional domains - boundary element - blocks spread of heterochromatin

50. Fedoriw 2004 Binding of CTCF is essential for proper H19/IGF2 imprinting