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京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17969号 / 生博第295号 / 新制||生||39(附属図書館) / 30799 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 米原 伸, 教授 河内 孝之, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24535号 / 医科博第149号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 江藤 浩之, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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ABSTRACT Retinoic acid (RA) is a positive regulator of P19 EC cell differentiation. Pre-B cell leukemia transcription factors (PBXs) act in conjunction with homeobox genes during cell differentiation. PBX mRNA and protein levels are increased rapidly in P19 cells during RA-induced differentiation. However, silencing of PBX expression in P19 cells (AS cells) results in a failure of these cells to differentiate upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) and Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) are orphan members of the steroid-thyroid hormone superfamily. The mRNA and protein levels of both COUP-TFI and COUP-TFII are low in proliferating wild type P19 EC cells. However, when wild type P19 cells are induced to differentiate upon RA treatment, COUP-TFI and COUP-TFII mRNA and protein levels are dramatically increased while the levels of pluripotency associated gene products are strikingly reduced. Conversely, COUP-TFI and COUP-TFII mRNA levels fail to be elevated upon RA treatment in PBX AS P19 EC cells. Therefore it was hypothesized that COUP-TFs may be downstream targets of PBX and required factors mediating the RA-dependent differentiation cascade in P19 cells. To determine the role of COUP-TFI during differentiation of P19 cells, PBX AS cells that inducibly express V5 tagged COUP-TFI using the Tet-Off® Advanced Inducible Gene Expression system were prepared. Using this system, we demonstrate that exogenous COUP-TFI expression, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption and early apoptosis. Furthermore, using this cell model which inherently is incapable of undergoing RA-mediated differentiation due to blockage of PBX induction, we demonstrate that a supraphysiological level of COUP-TFI expression can overcome the blockage of RA-dependent differentiation in PBX AS cells. However, AS cells expressing a physiological level of COUP-TFI differentiate to endodermal cells only upon treatment with RA. Additionally, gene expression studies indicate that the reductions of pluripotency maintenance genes observed in the COUP-TFI expressing cells are similar to that of wild type P19 cells (upon RA treatment) suggesting that COUP-TFI expression is a driving force towards loss of pluripotency. Moreover, gene expression studies indicate COUP-TFI is involved in the regulatory modulation of at least two RA response genes, CYP26A1 and HoxA1, indicating that COUP-TFI may have some effect on either maintaining or reducing these genes expression levels when COUP-TFI becomes expressed. COUP-TFII is expressed as two distinct variants, Variant 1(V1) and Variant 2 (V2). V1 is the variant that functions as a classical nuclear receptor by binding target DNA sequences and affecting gene transcription whereas V2 is a truncated form of V1 lacking the ability to bind DNA. We therefore hypothesized that V2 could serve as a dominant negative receptor by limiting the amount of functional V1 in the cell. Unexpectedly, we found using P19 cells that overexpress V2 that RA-mediated differentiation proceeded normally suggesting V2 does not function as a dominant negative repressor. Taken together, these studies demonstrate for the first time (i) that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells and (ii) COUP-TFII V2 is endogenously expressed in P19 cells; however its role during RA-mediated differentiation remains unclear. / Biochemistry

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