MOLECULAR PATHWAYS OF PLURIPOTENCY Dr. Serdar Sivgin February 2010 Kayseri 1

What is pluripotency At the blastocyst (day 5 after fertilization): An outer layer of cells, the trophectoderm (TE) A group of pluripotent cells, the ICM (inner cell mass). TE will develop into placental tissues TE will develop into placental tissues ICM gives rise to all cells of the embryo proper as well as several extraembryonic tissues. ICM gives rise to all cells of the embryo proper as well as several extraembryonic tissues. ICM and embryonic stem (ES) cells, possess the remarkable property of PLURİPOTENCY, the ability to give rise to all cells of the organism. ICM and embryonic stem (ES) cells, possess the remarkable property of PLURİPOTENCY, the ability to give rise to all cells of the organism. 2

Key transcription factors in pluripotency Key transcription factors such as Oct4, Sox2 or Nanog: *affect the cell cycle *regulate gene expression *modulate the epigenetic state *repair DNA damage Resulting in: Resulting in: *regulate PLURİPOTENCY. *functionally induce PLURİPOTENCY Besides transcription factors, microRNAs have recently been shown to play important roles in gene expression Besides transcription factors, microRNAs have recently been shown to play important roles in gene expression 3

Molecular mechanisms and key factors regulating the specification of ICM and TE lineages At the morula stage, cells choose their fate depending on their position and polarity. Genetic, epigenetic and environmental factors play an important role in early cell-fate Yap, the co-activator for transcription factor Tead4 (Yap localises in the nucleus and increases Tead4 activity) Tead4 subsequently activates the trophectoderm (TE) master factor Cdx2 Embryos lacking either Tead4 or Cdx2 fail to produce functional trophectodermal tissue but ICM cells remain intact and ES cells can be derived The counter-activity between Oct4 and Cdx2 allows the segregation of the first two embryonic lineages The counter-activity between Oct4 and Cdx2 allows the segregation of the first two embryonic lineages 4

Oct4 (octamer-binding transcription factor) Oocytes, fertilized embryo, embryonic carcinoma cells Oocytes, fertilized embryo, embryonic carcinoma cells The expression of Oct4 was detected in TE as well as ICM cells The expression of Oct4 was detected in TE as well as ICM cells Loss of Oct4: There is inappropriate differentiation of the inner cell mass and ES cells. So, ES cells cannot be derived of blastocyst So, ES cells cannot be derived of blastocyst Overexpression of Oct4: There is differentiation into primitive endoderm and mesoderm Oct4 can regulate gene expression by interacting with other factors within the nucleus, including the high mobility group (HMG)-box transcription factor Sox2 5

Nanog Morula, ICM, germ cells, embryonic carcinoma cells Required for the germline formation Required for the germline formation Cells lacking Nanog spontaneously differentiate into primitive endoderm Overexpression of Nanog promotes self-renewal independent of the cytokine leukemia inhibitory factor (LIF) Overexpression of Nanog promotes self-renewal independent of the cytokine leukemia inhibitory factor (LIF) Human and monkey ES cells seem to maintain the pluripotency in LIF/STAT3 independent manner 6

Sox2 (sex determining region Y)-box 2) Oocytes, ICM, epiblast,gut endoderm Oocytes, ICM, epiblast,gut endoderm Sox2 plays an important role in the maintenance of pluripotency and lineage specification. Sox2 plays an important role in the maintenance of pluripotency and lineage specification. * may be found in early neural stages. * may be found in early neural stages. * One of the earliest expressed genes for pluripotency. * One of the earliest expressed genes for pluripotency. 7

Gata4 and Gata6 * found in extraembryonic endoderm lineages * work as transcription factors. * work as transcription factors. *Forced expression of Gata4 or Gata6 in ES cells leads to differentiation into primitive endoderm, an effect similar to that caused by the loss of Nanog function *Gata4 and Gata6 expression was upregulated in the absence of Nanog 8

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Bone Morphogenetic Proteins (BMP) BMP are members of TGF-β superfamily BMP are members of TGF-β superfamily Receptors of the TGF-β of ligands Receptors of the TGF-β of ligands consist of a heteromeric complex of type I and type II receptor serine/ threonine kinases. consist of a heteromeric complex of type I and type II receptor serine/ threonine kinases. Binding of BMP to the receptor induces phosphorylation of R-Smads by type I receptors. Binding of BMP to the receptor induces phosphorylation of R-Smads by type I receptors. Phosphorylated R-Smads form complexes with Co- Smad and accumulate in the nucleus, where together they regulate gene transcription. Phosphorylated R-Smads form complexes with Co- Smad and accumulate in the nucleus, where together they regulate gene transcription. In human ES cells, several groups reported that BMP4 induces DIFFERENTIATION. In human ES cells, several groups reported that BMP4 induces DIFFERENTIATION. In mouse ES cells, BMP4 can induce expression of id (inhibitor of diffrerantiation) and suppress neural differentiation. In mouse ES cells, BMP4 can induce expression of id (inhibitor of diffrerantiation) and suppress neural differentiation. The self-renewal of mouse ES cells is achieved by a delicate balance between the two cytokines, LIF and BMP. The self-renewal of mouse ES cells is achieved by a delicate balance between the two cytokines, LIF and BMP. 10

Leukemia Inhibitory Factor (LIF) LIF is a heteromeric complex consisting of gp130 and the LIF receptor LIF is a heteromeric complex consisting of gp130 and the LIF receptor Upon LIF binding, JAK(Janus kinase) kinase phosphorylates tyrosine residues of both gp130 and LIFR. Upon LIF binding, JAK(Janus kinase) kinase phosphorylates tyrosine residues of both gp130 and LIFR. These phosphorylation recruits signal transducers and activators of transcription STAT 1 and STAT3 These phosphorylation recruits signal transducers and activators of transcription STAT 1 and STAT3 The activated STAT ( Signal transducer and activator of transcription 3) proteins translocate into the nucleus, where they function as transcription factors The activated STAT ( Signal transducer and activator of transcription 3) proteins translocate into the nucleus, where they function as transcription factors LIF and its downstream effector STAT3 are essential for maintenance of PLURİPOTENCY in mouse ES cells. LIF and its downstream effector STAT3 are essential for maintenance of PLURİPOTENCY in mouse ES cells. Human and monkey ES cells seem to maintain the pluripotency in LIF/STAT3 independent manner Human and monkey ES cells seem to maintain the pluripotency in LIF/STAT3 independent manner 11

Wnt/ β-catenin pathway The wingless gene had originally been identified as a recessive mutation affecting wing and haltere development in Drosophila melanogaster[3 ] Drosophila melanogaster[3 ]Drosophila melanogaster[3 ] Βeta-catenin is a cytoplasmic protein that functions in cell-cell adhesion by linking cadherins to the actin cytoskeleton. Βeta-catenin is a cytoplasmic protein that functions in cell-cell adhesion by linking cadherins to the actin cytoskeleton. In the absence of Wnt(combination of Wg (wingless ) and Int) activation, beta-catenin is phosphorylated by a complex consisting of APC gene, Axin, and glycogen synthase kinase (GSK) 3b. Phosphorylated beta-catenin is degraded by the ubiquitin proteasome system, thereby keeping the level of cytoplasmic beta-catenin low. Neural differentiation of mouse ES cells was attenuated by the activation of Wnt signaling by overexpression of Wnt1 or treatment with lithium chloride, an inhibitor of GSK3b 12

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Wnt/ β-catenin pathway may promote SELF-RENEWAL (in mouse and human ES cells) Wnt/ β-catenin pathway may promote SELF-RENEWAL (in mouse and human ES cells) Wnt binds to its receptor (Frizzled, LRP5 or LRP6) Wnt binds to its receptor (Frizzled, LRP5 or LRP6) Activated Dishevelled inactivates the APC/ Axin/ GSK3b complex. Activated Dishevelled inactivates the APC/ Axin/ GSK3b complex. Since this complex induces degradation of β-catenin in the absence of Wnt ligand, its inactivation results in the stabilization and accumulation of β-catenin protein in the nucleus. Since this complex induces degradation of β-catenin in the absence of Wnt ligand, its inactivation results in the stabilization and accumulation of β-catenin protein in the nucleus. Β-catenin binds to and activates LEF/TCF transcription factors. Β-catenin binds to and activates LEF/TCF transcription factors. 14

PI3 kinases are lipid kinases that catalyze the phosphorylation of inositol phospholipids PI3 kinase pathway is likely to be a crucial regulator of ES cell proliferation. PI3 kinase pathway may be involved in the maintenance of pluripotency in both mouse and human ES cells PI3 kinase inhibitor, suppressed progression of cells from the G1 to S phase and decreased cell proliferation PTEN is a negative regulator of the PI3 kinase pathway. In loss of negative regulations of PTEN promotes ES cell proliferation and tumorigenicity Phosphatidyl inositol 3 (PI3) kinase 15

Activation of the Ras/ERK pathway and PI3 kinase pathway by growth factors. The PI3 kinase pathway can be activated via different routes. Gab1 can bind to Grb2, resulting in tyrosine phosphorylation and activation of the PI3 kinase pathway. The PI3 kinase-regulatory subunit p85 can bind to a phosphorylated tyrosine residue of the receptor. Activated Ras can induce membrane localization and activation of the p110 catalytic subunit of PI3 kinase. The PI3 kinase pathway is constitutively activated by ERas in mouse ES cells. The PI3 kinase pathway can promote self-renewal of mouse and human ES cells, possibly by suppression of the ERK pathway 16

Phosphatidyl inositol 3 (PI3) kinase Activation of PI3 kinases is induced by many different receptor tyrosine kinases for growth factors, such as FGF, EGF, and PDGF, and leads to PIP3 Akt1 is a serine/threonine kinase. Akt1 binds to PIP3 and is translocated to the inner cell membrane, where it is phosphorylated and activated by another serine/threonine kinase PDK1 Activated Akt1 modulates the function of numerous substrates, such as Mdm2, IKK, and mTOR, and elicits various cellular responses, including proliferation and suppression of cell death. Activated Akt1 modulates the function of numerous substrates, such as Mdm2, IKK, and mTOR, and elicits various cellular responses, including proliferation and suppression of cell death. (everolimus, sirolimus mTOR inhibitors in RCC and AML) (everolimus, sirolimus mTOR inhibitors in RCC and AML) 17

Akt signaling pathway 18

Activation of the Ras/ERK pathway and PI3 kinase pathway by growth factors Binding of growth factors to their receptors induces autophosphorylation of receptors and/or phosphorylation of receptor-associated proteins. The adaptor protein Grb2 binds to the phosphorylated tyrosines through its SH2 domains and activates the Ras/ERK pathway through the GTP-GDP exchange factor SOS. Activation of the Ras/ERK pathway induces differentiation in mouse ES cells. 19

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Molecular mechanisms of reprogramming Re-establishing pluripotency in a somatic cell is a complicated process. The most important changes include the activation of an ES-cell-specific transcription network; *re-setting the epigenetic landscape *alteration of the cell cycle signature *overcoming the DNA damage response triggered by these drastic changes 21

Induced pluripotency with key factors ES cell factors such as Oct4, Sox2, cMyc, and Klf4 in fibroblast cells can reprogram them to a pluripotent state. The most efficient method to make iPS cells is through viral transduction. Failure of silencing indicates incomplete reprogramming and raises the danger of carcinogenesis by the oncogene cMyc. To avoid insertional mutagenesis and transgene reactivation, other methods that do not alter the genome have been developed, such as non-integrating episomal vectors, episomal vectors, minicircle vectors and minicircle vectors and PiggyBac transposon system PiggyBac transposon system 22

Differences between mouse and human ES cell The stem cells of teratocarcinoma are embryonal carcinoma (EC) cells, which express characteristics, similar to those of the inner cell mass (ICM) There are significant differences between mouse and human cells (EC and ES) Cell surface antigens of mouse EC and ES cells: SSEA1(+)/SSEA3(-)/SSEA4(-) Cell surface antigens of Human EC cells: Cell surface antigens of Human EC cells:SSEA1(-)/SSEA3(+)/SSEA4(+) (these phenotype is similar to that of human ES cells and human ICM cells) Human EC and ES cells have capacity to generate trophoblastic cells. This does not usually occur in mouse EC and ES cells. This does not usually occur in mouse EC and ES cells. 23

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Similarities and differences between mouse and human ES cell genomic targets Heart and neural crest derivatives expressed 1 (Hand1) and Myst3 genes were identified as targets of Oct4 and Nanog in human ES cells, whereas others such as Estrogen-related receptor b (Esrrb) were observed only in mouse cells whereas others such as Estrogen-related receptor b (Esrrb) were observed only in mouse cells Rif1 has been implicated in regulating telomere length and might be important for self-renewal Esrrb has been shown to be important for placental development and germ cell proliferation. Tcl1 is highly expressed in mouse ES cells, enhances cell proliferation and survival through augmentation of phosphoinositide-3 kinase PI3K–Akt signaling 26

Core transcriptional regulatory circuitry in pluripotent mouse and human ES cells. 27

Epigenetic control of pluripotency What is epigenetic? Each of the cells within our body contains the same genetic material, yet these cells can look and behave very differently Each cell contains the same genes but some are switched on (expressed) and some are switched off (not expressed).. The specific complement of genes expressed and not expressed in a cell determines its characteristics and this is controlled by epigenetics. ES cell chromatin characteristics: abundance of acetylated histone modifications increased accessibility to nucleases. 28

ES cells lacking Eed can contribute to most cell lineages, suggesting that PcG proteins are not necessary for maintaining pluripotency Eed mutant ES cells spontaneously differentiate PcG proteins are necessary for ES cell identity Gene expression is influenced by enzymatic activities that can induce both global and local changes in chromatin structure Gene expression is influenced by enzymatic activities that can induce both global and local changes in chromatin structure Epigenetic characteristics of pluripotent and lineage committed cells 29

Epigenetic characteristics of pluripotent and lineage committed cells. 30

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