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Lecture 5

Lecture 5. Differentiation and Reprogramming. You should understand;. Mechanisms that contribute to determination and maintenance of differentiated cell fates. Reprogramming occurs in the germ line and in early embryos.

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Lecture 5

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  1. Lecture 5 Differentiation and Reprogramming You should understand; • Mechanisms that contribute to determination and maintenance of differentiated cell fates. • Reprogramming occurs in the germ line and in early embryos • There are several experimental strategies to reprogram differentiated cells • Unique features of the pluripotent state

  2. Differentiation and reprogramming - overview Embryonic progenitor/ES cell Adult stem cell Differentiated cells • 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. • Adult stem cells retain some degree of plasticity. • Interconversion of differentiated cells = transdifferentiation (red dashed line) • Reversal of differentiation back to embryonic state = reprogramming (blue dashed line).

  3. Cell identity - ‘memory’ mechanisms Embryonic progenitor/ES cell Adult stem cell Differentiated cells • Cell identity = the sum of ‘on’ vs ‘off’ genes - generally stable • Transcriptional circuits stabilised by feedback mechanisms • Epigenetic mechanisms increase the stability of cell identity

  4. 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, egSox2/Oct4/Nanogin ES cells and Cdx2/Gata3 in trophectoderm.

  5. Memory mechanisms; 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. 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

  7. Heritable gene silencing by CpG DNA methylation Me CpG GpC Me • Methylation patterns are established by Dnmt3a/b • in early development. • Faithfully maintained through DNA replication • (Dnmt1). • Repressive but limited role in gene regulation; imprinted • genes, inactive X chromosome, Nanog and other • pluripotency genes in early zygote and somatic cells. • Oct4 in developing embryo.

  8. 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

  9. 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

  10. 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. • In mammals reprogramming is part of normal development, specifically in • developing germ cells and in preimplantation embryos. • Experimental reprogramming in mammalian cells achieved by cloning (Dolly) but also by cell • fusion, and more recently using iPS technology. Briggs and King (1952) Proc Natl Acad Sci U S A. 38, p55-63; Gurdon et al (1958) Nature 182, p64-5

  11. Reprogramming during germ cell development Pre-natal • Repression of somatic program and reactivation pluripotencyprogram • 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).

  12. Reprogramming in preimplantation development TET proteins (TET1/2/3) are DNA hydroxylases that oxidise 5-methyl cytosine. Wu and Zhang, (2011) Genes and Dev. 25, p2436-2452, Review. • 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. • Reactivation of inactive X chromosome in ICM cells.

  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 – • Cells are reprogrammed back to a totipotent state • Frequency of success (liveborn) remains poor, less than 1/100. • Cloning of a mouse from a lymphocyte 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 Jaenisch (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 • Mouse ES cell rapidly activates ES cell program in human • B-lymphocyte genome in transient heterokaryon. • Precocious DNA synthesis induced in the somatic nucleus • is required for reprorgramming. • Pereira et al (2008). PLoS Genet. 4, e1000170 • Tsubouchi et al (2013) Cell 152, p873–883.

  17. Fbx15 Nanog etc Fbx15 Nanog etc X Neomycin resistance ORF Neomycin resistance ORF 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 • Conversion occurs without c-myc but less efficiently – cell cycle effects?

  19. Stimulus Triggered Acquisition of pluripotency (STAP) cells Obokata et al(2014) Nature 505, p641-647 • Low pH triggers conversion of somatic cells to pluripotency. Increased Oct4, decreased DNA methylation etc • Contribute to all lineages, including trophectoderm • Some controversy!

  20. What is unique about the pluripotent state? Examples of trans-differentiation; • Forced MyoD expression can convert a variety of cell types into myoblasts • 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

  21. What is unique about 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/reverse epigenetic information in somatic cells • e.gDNA and histone demethylases. • Disengagement of epigenetic feedback loops that stabilise transcription on/off switches • In somatic cells. Azuara et al (2006) Nat Cell Biol. 8, p532-8; Bernstein et al (2006) Cell 125, p315-26

  22. The application of reprogramming technology • Similar to human ES cells – not ground state (yet) • Human iPS cells derived from fibroblasts using Yamanaka factor cocktails. • Potential application as patient specific stem cells for regenerative medicine Thomson et al (1998) Science 282, p1145-7

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

  24. End lecture 5

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