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OCT4 activity during conversion of human intermediately reprogrammed stem cells to iPSCs through mesenchymal-epithelial transition / 間葉上皮転換を経てヒト再プログラム化中間細胞がiPS細胞へ再プログラム化する過程でのOCT4遺伝子活性Teshigawara, Rika 25 March 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21655号 / 医博第4461号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 江藤 浩之, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Pou5f1 Post-translational Modifications Modulate Gene Expression and Cell FateCampbell, Pearl 20 December 2012 (has links)
Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.
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Pou5f1 Post-translational Modifications Modulate Gene Expression and Cell FateCampbell, Pearl 20 December 2012 (has links)
Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.
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Apical Ectodermal Ridge (AER) activity and limb outgrowth during vertebrate development11Viegas Tomás, Ana Raquel 11 January 2011 (has links)
Limb outgrowth is controlled by a specialized group of cells called the apical ectodermal ridge (AER), a
thickening of the limb epithelium, at its distal tip. This specialized thickening of ectodermal cells is
responsible for maintaining the underlying mesenchymal cells in an undifferentiated and proliferative state,
and its structure is preserved through a fine-tuned balance between proliferation and apoptosis. This
equilibrium is genetically controlled but little is known about the molecules involved in this process. Several
authors have been shown that both fibroblast growth factor (FGF) and Erk pathway activation are crucial for
AER function. Recently, FLRT3, a transmembrane protein able to interact with FGF receptors, has been
implicated in the triggering of ERK activity by FGFs. In this thesis, we show that flrt3 expression is
restricted to the AER, co-localizing its expression with fgf8 and pERK activity. Loss-of-function studies
demonstrate that silencing of flrt3 affects the integrity of the AER and, subsequently, its proper function
during limb bud outgrowth. Our data also indicate that flrt3 expression is not regulated by FGF activity in
the AER, whereas ectopic WNT3A is able to induce flrt3 expression. Overall, our findings confirm flrt3 as a
key player during chicken limb development, being necessary but not sufficient for proper AER formation
and maintenance under the control of BMP and WNT signalling.
During limb bud development, AER structure is maintained through a fine-tuned balance between
proliferation and programmed cell death and this equilibrium is genetically controlled, although little is
known about the molecules involved in that process.
In this thesis we present evidences involving oct4, required to establish and maintain the pluripotent cell
population necessary for embryogenesis in mouse and human, in the control of the proliferative balance
within the AER cells. Overexpression of otc4 in the limb ectoderm disrupts the ratio apoptosis/proliferation
and, moreover, oct4 expression is under the control of wnt-canonical pathway. We also describe a special
localization and behaviour of proliferating cells in the AER in response to oct4 activity. We, therefore,
describe a role for oct4 as a factor able to maintain a niche of cells that is responsible for the renewal of the
AER. / El crecimiento del esbozo de la extremidad está controlado por un grupo especializado de células
denominado Cresta Ectodérmica Apical (CEA), un engrosamiento del epitelio del miembro en su borde más
distal. Este engrosamiento es responsable del mantenimiento de las células del mesodermo distal en un
estado indiferenciado y proliferativo. Diferentes estudios muestran que la actividad de los factores de
crecimiento fibroblástico (FCF) y de la vía Erk son cruciales para la correcta funcionalidad de la CEA.
Recientemente se ha implicado a FLRT3, una proteína transmembranal capaz de interaccionar con los
receptores de los FCF, en la activación de la vía Erk por los mismos. En esta tesis describimos cómo la
expresión de flrt3 se restringe a la CEA, colocalizándose su expresión con fgf8 y la actividad de la vía Erk.
Los experimentos de pérdida de función demuestran que la inhibición de flrt3 afecta la integridad de la CEA
y, consecuentemente, a su función durante el desarrollo del esbozo del miembro. Nuestros datos también
indican que la expresión de flrt3 no está regulada a través de los FCF en la CEA, sin embargo, la activación
ectópica de WNT3A es capaz de inducir la expresión de flrt3. En conjunto, nuestros resultados demuestran
que flrt3 es una molécula clave durante el desarrollo de las extremidades de pollo, siendo necesaria, pero no
suficiente, para la correcta formación y mantenimiento de la CEA bajo el control de la señalización a través
de BMP y WNT.
Durante el desarrollo de las extremidades, la estructura de la CEA se mantiene a través de un fino control del
balance entre la proliferación y apoptosis. Este equilibrio se encuentra genéticamente controlado aunque se
sabe muy poco acerca de las moléculas involucradas en este proceso.
En esta tesis presentamos evidencias en las que oct4, molécula necesaria para establecer y mantener la
población de células pluripotentes necesarias durante la embriogénesis en ratón y humanos, controla la tasa
de proliferación en las células de la CEA. La expresión ectópica de oct4 en el ectodermo del esbozo de la
extremidad perturba la razón entre la apoptosis y la proliferación y, además, su expresión está controlada por
la actividad de la vía canónica de los Wnt. También describimos en este trabajo la localización y
comportamiento especiales de las células de la CEA en proliferación como respuesta a la actividad de oct4.
Por consiguiente, podemos inferir que el rol de oct4 será el de un factor necesario para mantener un nicho
celular responsable por la renovación de la CEA.
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Pou5f1 Post-translational Modifications Modulate Gene Expression and Cell FateCampbell, Pearl January 2012 (has links)
Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.
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