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

2. As development proceeds cell fate becomes progressively restricted and there is a loss of plasticity. Adult stem cells retain some degree of plasticity. Cells of the early embryo differentiate into many cell types – plasticity. Reversal of differentiation back to embryonic state = reprogramming (blue dashed line). Differentiation and reprogramming - overview Embryonic progenitor/ES cell Differentiated cells Adult stem cell Interconversion of differentiated cells = transdifferentiation (red dashed line)

3. Cell identity - ‘memory’ mechanisms 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 Embryonic progenitor/ES cell Differentiated cells Adult stem cell

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 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, eg Sox2/Oct4/Nanog in ES cells and Cdx2/Gata3 in trophectoderm. MyoD can induce a muscle specific expression program in several but not all cell types analysed.

5. Memory mechanisms; X inactivation and imprinting Inactive X chromosome Repressive chromatin marks Active X chromosome Imprinted gene silent on paternal chromosome Imprinted gene active on maternal chromosome Transcription factors/master regulators Nucleus

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

7. Heritable gene silencing by CpG DNA methylation 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. CpG GpC Me

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. Methylation of histone H3 lysine 27 Ubiquitylation of histone H2A lysine 119 ATP dependent chromatin remodelling Methylation of histone H3 lysine 4 or 36 Polycomb group Trithorax group Mechanism for stable propagation of histone marks not well understood

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

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

13. Cloning 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 Briggs and King and then Gurdon experiments demonstrated amphibian oocytes can induce complete reprogramming of a somatic cell nucleus. Cloning of a mouse from a lymphocyte proves cloning of terminally differentiated cell is possible. Frequency of success (liveborn) remains poor, less than 1/100. Campbell, Wilmut and colleagues, 1996 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 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 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 Cell cycle stage of donor nucleus influences efficiency (G1 or G0 thought to be best) - mechanism unknown In cloned mouse blastocysts activation of pluripotency genes is often incomplete and highly variable Factors influencing efficiency of cloning

15. Cell fusion of somatic and pluripotent cells 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. Cell type A Cell type B Sendai virus PEG Electroshock Heterokaryon 4N hybrid Same or different species 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 2N hybrid

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. Induced pluripotent stem (iPS) cells Mouse iPS cells contribute to chimeras and can be passed through the germline Neomycin resistance ORF Fbx15 Nanog etc X Fibroblast cells Neomycin resistance ORF Fbx15 Nanog etc iPS cells Introduce genes for ES cell factors X24 then narrowed down to; Oct4, Sox2, Klf4, c-myc + LIF + feeders + neomycin Approx 2 weeks….. Reactivation of somatic cell inactive X chromosome. iPS cells induce endogenous pluripotency genes and switch off fibroblast program. 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 Low pH triggers conversion of somatic cells to pluripotency. Increased Oct4, decreased DNA methylation etc Contribute to all lineages, including trophectoderm Some controversy! Obokata et al (2014) Nature 505, p641-647

21. What is unique about the pluripotent state? 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 Examples of trans-differentiation;

22. What is unique about the pluripotent state? Expression of factors required to erase/reverse epigenetic information in somatic cells e.g DNA and histone demethylases. 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. Azuara et al (2006) Nat Cell Biol. 8, p532-8; Bernstein et al (2006) Cell 125, p315-26 Disengagement of epigenetic feedback loops that stabilise transcription on/off switches In somatic cells.

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

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

25. End lecture 5