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Lecture 4

Lecture 4. Differentiation and Reprogramming Maintenance and stability of differentiated cell states. Reprogramming in normal development. Experimental Reprogramming. You should understand;. Mechanisms that contribute to determination and maintenance of differentiated cell fates.

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Lecture 4

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  1. Lecture 4 Differentiation and Reprogramming Maintenance and stability of differentiated cell states Reprogramming in normal development Experimental Reprogramming You should understand; Mechanisms that contribute to determination and maintenance of differentiated cell fates. Reprogramming in the germ line and in early embyros Experimental reprogramming approaches

  2. Differentiation and reprogramming - overview Who am I? • Cell identity is conferred by the transcriptional program, the sum of ‘on’ vs ‘off’ genes. • Cell identity is generally stable, attributable to ‘memory’ mechanisms. • Cells of the early embryo differentiate into many cell types – plasticity. • As development proceeds cell fate becomes progressively restricted and there is a loss of plasticity. • Stem cells retain some degree of plasticity. • Terminally differentiated cells are generally quiescent or divide slowly. • The identity of differentiated cells can be reversed back to a more plastic embryonic state in certain circumstances - reprogramming.

  3. Memory mechanisms; master transcription factors define cell type specific transcription programs Davis et al (1987) Cell 51, p987-1000 • MyoD, a muscle specific helix-loop-helix transcription factor converts fibroblast to myoblasts • when expressed from a heterologous promoter • MyoD can induce a muscle specific expression program in several but not all cell types • analysed. • MyoD cooperates with three related transcription factors, myf5, mrf4 and myogenin to promote • muscle identity • Myogenic transcription factors directly activate muscle specific genes, including themselves • and one another, forming an autoregulatory loop that stabilises muscle cell identity • Participation of master transcription factors in autoregulatory loops facillitates stabilisation of • cell identify in other cell types, egSox/Oct4/Nanog in ES cells and Cdx2 in trophectoderm.

  4. Chromatin modification contributes to maintenance of cell identity and ‘memory’ by creating stable (epigenetic/heritable) on and off states. Open/accessible/ permissive (active promoters, replication sites, repair sites) Closed/inaccessible/non-permissive (centromeres/telomeres, inactive X, silent promoters) Modifications and variants Writers Readers Lysine acetylation Bromodomain proteins HATs and HDACs Lysine methylation KHMTase and KDMase Chromodomain proteins Arginine methylation PRMTs and demethylases Tudor domain proteins Lysine ubiquitylation E3 ligases and DUBs MBD domain proteins Ser/Thr phosphorylation • Histone tail modifications (acetylation, methylation, phosphorylation, ubiquitylation etc) DNA (cytosine) methylation Kinases and phosphatases PHD, PWWP, ADD etc +Linker histone (H1) • Histone variants (H1 types, H2AZ, H2AX, CENPA, H3.1/3.3 etc) Dnmts and demethylases None of the above! + Histone variants (Cenp, H2AZ etc) • DNA methylation

  5. Allele specific chromatin silencing – X inactivation and imprinting Transcription factors/master regulators Nucleus Active X chromosome Inactive X chromosome Repressive chromatin marks Imprinted gene silent on paternal chromosome Imprinted gene active on maternal chromosome

  6. Heritable gene silencing by DNA methylation • Methylation patterns are established by Dnmt3a/b • in early development. • Faithfully maintained through DNA replication • (Dnmt1). • Limited function in gene regulation; imprinted • genes, inactive X chromosome, Nanog and other • pluripotency genes in early zygote and somatic cells.

  7. Polycomb and Trithorax proteins are ‘memory’ factors that stabilise cell identity • Genetic studies in fly identify factors required to maintain ‘on’ state (trithorax group/TrxG) or ‘off’ state • (Polycomb group/PcG) of hox cluster genes. • Highly conserved and important for regulation of developmental genes in all multicellular organisms. Simon and Kingston (2009) Nat Rev Mol Cell Biol 10, p697-708. Review

  8. PcG and TrxG proteins participate in multiprotein complexes that modify chromatin. Trithorax group Polycomb group Methylation of histone H3 lysine 27 Ubiquitylation of histone H2A lysine 119 ATP dependent chromatin remodelling Methylation of histone H3 lysine 4 or 36 • Mechanism for stable propagation of histone marks not well understood

  9. Reprogramming • Nuclear transfer experiment suggested by Spemann in 1938, was performed for blastocyst • cells by Briggs and King, 1952, and for tadpole and then adult cells by Gurdon, 1957. • Reprogramming in mammalian cells achieved by cell fusion, cloning (Dolly) and more • recently by iPS technology. • Reprogramming is part of normal development in mammals, specifically in • developing germ cells and in preimplantation embryos . Briggs and King (1952) Proc Natl Acad Sci U S A. 38, p55-63; Gurdon et al (1958) Nature 182, p64-5

  10. Reprogramming during germ cell development Pre-natal • Repression of somatic program and reactivation of potential pluripotency program • Changes in global histone modification status • Loss of DNA methylation (active/passive?) including erasure of parental imprints Post-natal • De novo DNA methylation including imprinted loci (different for male and female germ cells).

  11. Reprogramming DNA methylation in preimplantation development • Active (replication independent) and passive (replication linked) demethylation occur between 1-cell • and blastocyst stage. • Methylation is re-established by de novo Dnmts from blastocyst through to egg-cylinder stages. • Methylation of imprinting control regions is protected from genome wide demethylation.

  12. 10.5 X chromosome reactivation in primitive ectoderm of the ICM 2.5 0 1.5 4.5 6.0 3.5 Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm or • Xp Xist is switched on at the 2-cell stage and the paternal X chromosome (Xp) is inactivated in • all cells of cleavage stage embryos (imprinted X inactivation). Xp Xist ON Xp Inactivation Xp Reactivation Random Xist ON Random X inactivation • Thereafter Xp inactivation is stably maintained in trophectoderm and primitive endoderm lineage cells • Xp Xist is switched off and Xp is reactivated in primitive ectoderm cells of the ICM • Xp or Xm Xist is activated at random, leading to random X inactivation in epiblast cells of • Postimplantation blastocyst (E5.5). Mak et al (2004 ) Science;303, p666-9 ; Okamoto et al (2004) Science 303, p644-9

  13. Campbell, Wilmut and colleagues, 1996 Cloning • Briggs and King and then Gurdon experiments demonstrated amphibian oocytes can induce • complete reprogramming of a somatic cell nucleus. • Many failed attempts to clone mammals led to the belief this wouldn’t be possible until Dolly • Methodology now extended to mouse, cat, cow and many other mammalian species • Frequency of success (liveborn) remains poor, less than 1/100. • Cloning of a mouse from a lymphocyte finally proves cloning of terminally differentiated cell is possible. Campbell et al (1996) Nature 380, p64-6; Wakayama et al (1998), Nature 394, p369-74 ; Hochedlinger and Jaensch (2002) Nature 415, p1035-8

  14. Cloning Factors influencing efficiency of cloning • Cloned animals often have serious health problems with fetal overgrowth being commonplace – attributable to misexpression of important genes • Analysis of cloned mice indicate up to 4% of genes misexpressed • In cloned mouse blastocysts activation of pluripotency genes is often incomplete and highly variable • Cloned female mouse embryos partly reprogram X inactivation but efficiency of cloning much improved • in Xist knockout, both in male and female, suggesting that donor cell Xist is often inappropriately activated • Cell cycle stage of donor nucleus influences efficiency (G1 or G0 thought to be best) - mechanism unknown

  15. Cell fusion of somatic and pluripotent cells Sendai virus PEG Electroshock Cell type A Cell type B 4N hybrid 2N hybrid Heterokaryon Same or different species • Pioneering experiments by Henry Harris in 1969 demonstrated dominance - suppression of transformed • phenotype following fusion of transformed cells and certain normal cells – posited tumour supressor loci • Blau and colleagues demonstrated fibroblasts converted to myoblasts in myoblast/fibroblast fusion • Ruddle, Takagi, Martin and others show EC cell hybrids with somatic cells have pluripotent • differentiative capacity and reactivate inactive X chromosome. Harris et al (1969) J. Cell Sci. 4, p449-525; Blau et al (1985) Science 230, p758-766; Miller and Ruddle (1976) Cell 9, p45-55; Takagi et al (1983) Cell 34, p1053-62; Martin et al (1978) Nature 271, p329-33

  16. Cell fusion of somatic and pluripotent cells

  17. Fbx15 Nanog etc Fbx15 Nanog etc X Neomycin resistance ORF Neomycin resistance ORFf Induced pluripotent stem (iPS) cells Fibroblast cells iPS cells Introduce genes for ES cell factors X24 then narrowed down to; Oct4, Sox2, Klf4, c-myc + LIF + feeders + neomycin Approx 2 weeks….. • iPS cells induce endogenous pluripotency genes and switch off fibroblast program. • Mouse iPS cells contribute to chimeras and can be passed through the germline • Reactivation of somatic cell inactive X chromosome. Takahashi and Yamanaka (2006) Cell 126, p663-76

  18. Induced pluripotent stem (iPS) cells Conversion to iPS cells is relatively inefficient – why? • Requires sequential activation of different endogenous ES cell factors at different times – • stepwise reversal of differentiation? • Stochastic epigenetic changes

  19. Transdifferentiation by master transcription factors • Forced MyoD expression can convert a variety of cell types into myoblasts • B-cells to macrophage by addition of C/EBP • B-cells to macrophage by addition of C/EBP • Pancreatic exocrine to endocrine cells by Ngn3, Pdx1 and MafA cocktail. • Fibroblasts to neuron like cells by Ascl1, Brn2, and Mytl1 Hanna et al (2010) Cell 143, p508-525. Review

  20. Uniqueness of the pluripotent state Availability of unlimited quantity of ES cells grown in vitro has facillitated genome wide analysis. Key findings include; • Core transcription factors Oct4, Nanog and Sox2 co-occupy a large proportion of target genes • Oct4, Nanog and Sox2 participate in positive feedback loops with themselves and one another to stably maintain the pluripotent state • Oct, Nanog and Sox2 participate in negative regulatory loops to block expression of core transcription factors of trophectoderm and primitive endoderm lineages. • Other target genes can be either activated or repressed (recruitment of co-activators or co-repressors). • Repressed target genes are associated with differentiation into different lineages and are held in a‘poised’ configuration by epigenetic mechanisms (Polycomb). Boyer et al (2005) Cell 122, p947-56

  21. Uniqueness of the pluripotent state • Oct4/Nanog/Sox2 directly repress master regulators of many other lineages - • associated with presence of repressive together with active histone modifications • (bivalency), suggesting a poised state. • Expression of factors required to erase epigenetic information in somatic cells e.g DNA and • histone demethylases. • Disengagement of epigenetic feedback loops…… Azuara et al (2006) Nat Cell Biol. 8, p532-8; Bernstein et al (2006) Cell 125, p315-26

  22. on on on on off off off off Reversibility of X inactivation in ES cells • Disengagement of epigenetic feedback loops…… Normal differentiation/development on on Undifferentiated ES cell Active X chr on on on on Inactive X chr Xist RNA Wutz and Jaenisch (2000) Mol Cell. 5, p695-705

  23. The application of reprogramming technology • Human ES cell lines first isolated in 1998 • Derived from blastocyst stage embryos and grow indefinitely with stable karyotype. • Express ES cell markers such as alkaline phosphatase and coretranscription factors Nanog, Oct4 and Sox2’ in common with mouse ES cells. • Not LIF/BMP dependent - require FGF2 and Activin instead. • Have capacity to differentiate into cell types from all three germ layers (+ trophectoderm) – potential use in regenerative medicine. • Human iPS cells derived from fibroblasts using Yamanaka factor cocktails. Thomson et al (1998) Science 282, p1145-7

  24. The application of reprogramming technology • Cell/tissue replacement • Disease models (patient specific cell lines) • Drug testing • Cell factories Challenges; • Heterogeneity in iPS lines/incomplete reprogramming • Teratoma formation See Yamanaka and Blau review

  25. End lecture 4

  26. Reading list Textbook;  Principles of Development, Lewis Wolpert and Cheryl Tickle. Review papers; Lecture 1 and 2 Alexandre (2001) International Journal of Developmental Biology 45, p457-467 Rossant (2001) Stem Cells 19, p477-82 Yamanaka et al, (2006). Developmental Dynamics 235, p2301-2314 Katsuyoshi and Hamada, (2012) Development 139, p3-14 Lecture 3 and 4 Arnold and Robertson (2009) Nature reviews Molecular cellular biology, 10, p91-103 Robb and Tam (2004) Seminars in Cell and Developmental biology 15, p43-54 Hayashi et al (2007) Science 316, p394-396. Hanna et al (2010) Cell 143, p508-525. Yamanaka and Blau (2010) Nature 465, p704-712

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