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
Differentiation and reprogramming - overview
Stem cell; unlimited capacity to self-renew
and produces differentiated derivatives
Progenitor cell; limited capacity to self-renew
and can produce differentiated derivatives
Terminally differentiated/specialised cell
• 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.
• Adult stem cells retain some degree of plasticity.
• The identity of differentiated cells can be reversed back to a more plastic embryonic state in
certain circumstances - reprogramming.
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, eg Sox2/Oct4/Nanog in ES cells and Cdx2/Gata3 in trophectoderm.
Chromatin modification contributes to maintenance of
cell identity and ‘memory’ by creating
stable (epigenetic/heritable) on and off states.
Open/accessible/permissive
Closed/inaccessible/non-permissive
(active promoters, replication sites, repair sites)
(centromeres/telomeres, inactive X, silent promoters)
Modifications and variants
Lysine acetylation
Lysine methylation
Arginine methylation
Lysine ubiquitylation
Ser/Thr phosphorylation
DNA (cytosine) methylation
+
+
Linker histone (H1)
Histone variants
(Cenp, H2AZ etc)
Writers
Readers
HATs and HDACs
Bromodomain proteins
KHMTase and KDMase
Chromodomain proteins
PRMTs and demethylases
Tudor domain proteins
E3 ligases and DUBs
MBD domain proteins
Kinases and phosphatases
PHD, PWWP, ADD etc
Dnmts and demethylases
None of the above!
•
Histone tail modifications (acetylation, methylation, phosphorylation, ubiquitylation etc)
•
Histone variants (H1 types, H2AZ, H2AX, CENPA, H3.1/3.3 etc)
•
DNA methylation
Memory mechanisms; chromatin effects
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
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.
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
PcG and TrxG proteins participate in multiprotein complexes
that modify chromatin.
Polycomb group
Methylation of
histone
H3 lysine 27
•
Ubiquitylation of
histone
H2A lysine 119
Trithorax group
ATP dependent
chromatin
remodelling
Methylation of
histone
H3 lysine 4 or 36
Mechanism for stable propagation of histone marks not well understood
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
Reprogramming during germ cell development
Pre-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
Post-natal
•
De novo DNA methylation including imprinted loci (different for male and female germ cells).
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.
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
Campbell, Wilmut and colleagues, 1996
• 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 Jaenisch (2002) Nature 415, p1035-8
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
Cell fusion of somatic and pluripotent cells
Cell type A
Sendai virus
PEG
Electroshock
Cell type B
Heterokaryon
4N hybrid
2N hybrid
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
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.
Induced pluripotent stem (iPS) cells
Fibroblast cells
Introduce genes for ES cell factors
X24 then narrowed down to;
Oct4, Sox2, Klf4, c-myc
iPS cells
+ LIF + feeders + neomycin
Approx 2 weeks…..
X
Fbx15
Nanog
etc
Neomycin resistance ORF
Fbx15
Nanog
etc
Neomycin resistance ORF
• 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
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?
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
• 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
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
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 core transcription 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
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
End lecture 4