Lecture 4

Differentiation and Reprogramming

Maintenance and stability of differentiated cell states

Reprogramming in normal development

Experimental Reprogramming

You should understand;

Reprogramming in the germ line and in early embyros

Experimental reprogramming approaches

Mechanisms that contribute to determination and maintenance of differentiated cell fates.

Who am I?

• As development proceeds cell fate becomes progressively restricted and there is a loss of plasticity.

• Stem cells retain some degree of plasticity.

• 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.

• The identity of differentiated cells can be reversed back to a more plastic embryonic state in certain circumstances - reprogramming.

Differentiation and reprogramming - overview

• Terminally differentiated cells are generally quiescent or divide slowly.

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 myoblastswhen expressed from a heterologous promoter

• MyoD cooperates with three related transcription factors, myf5, mrf4 and myogenin to promotemuscle identity

• Myogenic transcription factors directly activate muscle specific genes, including themselvesand one another, forming an autoregulatory loop that stabilises muscle cell identity

• Participation of master transcription factors in autoregulatory loops facillitates stabilisation ofcell identify in other cell types, eg Sox/Oct4/Nanog in ES cells and Cdx2 in trophectoderm.

• MyoD can induce a muscle specific expression program in several but not all cell typesanalysed.

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

Allele specific chromatin silencing – X inactivation and imprinting

Inactive Xchromosome

Repressivechromatin marks

Active Xchromosome

Imprinted genesilent on paternal chromosome

Imprinted geneactive on maternal chromosome

Transcription factors/master regulatorsNucleus

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; imprintedgenes, inactive X chromosome, Nanog and otherpluripotency genes in early zygote and somatic cells.

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.

Methylation of histoneH3 lysine 27

Ubiquitylation of histoneH2A lysine 119

ATP dependentchromatin remodelling

Methylation of histoneH3 lysine 4 or 36

Polycomb group Trithorax group

• Mechanism for stable propagation of histone marks not well understood

Reprogramming

• Reprogramming is part of normal development in mammals, specifically in developing germ cells and in preimplantation embryos .

• Reprogramming in mammalian cells achieved by cell fusion, cloning (Dolly) and morerecently by 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

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 of potential pluripotency program

• Changes in global histone modification status

• Loss of DNA methylation (active/passive?) including erasure of parental imprints

Pre-natal

Reprogramming DNA methylation in preimplantation development

• Active (replication independent) and passive (replication linked) demethylation occur between 1-celland 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.

• 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).

X chromosome reactivation in primitive ectoderm of the ICM

Xp Xm

0 3.5 10.5

Xp Xm

1.5

Xp Xist

ON

Xp Xm

2.5

XpInactivation

Xp Xm

4.5

XpReactivation

or

Xp Xm Xp Xm

6.0

Random Xist ONRandom 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

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

• 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 finally 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 Jaensch (2002) Nature 415, p1035-8

Cloning

• Cloned female mouse embryos partly reprogram X inactivation but efficiency of cloning much improvedin 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

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 pluripotentdifferentiative capacity and reactivate inactive X chromosome.

Cell type A

Cell type B

Sendai virusPEGElectroshock

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

Cell fusion of somatic and pluripotent cells

Induced pluripotent stem (iPS) cells

• Mouse iPS cells contribute to chimeras and can be passed through the germline

Neomycin resistance ORF

Fbx15NanogetcX

Fibroblast cells

Neomycin resistance ORFf

Fbx15Nanogetc

iPS cellsIntroduce genes for ES cell factorsX24 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

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

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

Uniqueness of the pluripotent state

Availability of unlimited quantity of ES cells grown in vitro has facillitated genome wideanalysis. Key findings include;

• Oct, Nanog and Sox2 participate in negative regulatory loops to block expression of core transcription factors of trophectoderm and primitive endoderm lineages.

• Core transcription factors Oct4, Nanog and Sox2 co-occupy a large proportion of target genes

• 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).

• Oct4, Nanog and Sox2 participate in positive feedback loops with themselves and one another to stably maintain the pluripotent state

Boyer et al (2005) Cell 122, p947-56

Uniqueness of the pluripotent state

• Expression of factors required to erase 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.

• Disengagement of epigenetic feedback loops……

Azuara et al (2006) Nat Cell Biol. 8, p532-8; Bernstein et al (2006) Cell 125, p315-26

Undifferentiated ES cell

on on

Normal differentiation/development

Reversibility of X inactivation in ES cells• Disengagement of epigenetic feedback loops……

Wutz and Jaenisch (2000) Mol Cell. 5, p695-705

on off on off on off

on on on off on onActive X chr

Inactive X chr

Xist RNA

• Human ES cell lines first isolated in 1998

• Derived from blastocyst stage embryos and grow indefinitely with stable karyotype.

• 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.

• Express ES cell markers such as alkaline phosphatase and core transcription factors Nanog, Oct4 and Sox2’ in common with mouse ES cells.

Thomson et al (1998) Science 282, p1145-7

The application of reprogramming technology

• Human iPS cells derived from fibroblasts using Yamanaka factor cocktails.

The application of reprogramming technology

• Cell/tissue replacement

• Disease models (patient specific cell lines)

• Cell factories

• Drug testing

Challenges;

• Teratoma formation

• Heterogeneity in iPS lines/incomplete reprogramming

See Yamanaka and Blau review

End lecture 4

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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