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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18141号 / 医博第3861号 / 新制||医||1002(附属図書館) / 30999 / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 髙田 穣, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1

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<p>Recent advances in gene targeting technology have significantly shaped modern-day mouse genetics, as they allow for the accurate analysis of gene function <em>in vivo</em>. By capitalizing on conventional methodologies that are based on homologous recombination, the advent of artificially engineered nucleases, like transcription activator-like effector nucleases (TALENs), enables precise genome editing without the need for conventional targeting vectors, which typically possess long “arms” of homology that are difficult to work with, even with recombineering strategies employing bacterial artificial chromosomes. Unlike traditional techniques, these novel nucleases can be engineered in less than a week and together with compact targeting vectors, can be used to easily manipulate almost any locus in the mouse genome.</p> <p>The current selection of commercially available antibodies makes it difficult to assess the specific roles of protein isoforms during early development. The Tcf/Lef family of transcription factors comprise of key downstream effector proteins of the canonical Wnt/β-catenin signal transduction cascade. This pathway is implicated in the regulation of self-renewal and is dysregulated in a number of human diseases including cancers. Among the Tcf/Lef factors, Tcf3 has been heavily studied in mouse embryonic stem cells, due at least in part to the observation that its transcript levels are expressed at the highest levels compared to the others. Recently, it was proposed that a switch takes place between a repressive state mediated by Tcf3 to an activating β-catenin-Tcf1 complex in response to Wnt signals. Here, we use TALEN technology to introduce an epitope tag at the endogenous locus of <em>mTcf7</em>, the gene encoding the Tcf1 protein. By tagging the N-terminus of full-length and N-terminally truncated dominant-negative variants of Tcf1, we establish a tool to better study a previously unappreciated role for Tcf1 in regulating embryonic stem cell self-renewal and differentiation. Furthermore, we also show that the tagged variants generated exhibit similar protein expression levels to those of wild-type controls, and display nuclear localization as expected.</p> / Master of Science (MSc)

The nuclear pore complex is closely involved in the regulation and control of many cellular processes, including the movement of molecules into and out of the nucleus along with the regulation of gene transcription. It is therefore a major barrier for controlling the passage of signaling molecules into and out of the nucleus. β-catenin is one such signaling molecule, and a primary signaling molecule of the Wnt signaling pathway. How the passage of β-catenin into and out of the nucleus is controlled remains poorly defined. This signaling pathway governs major developmental processes, including cell fate determination, proliferation, motility and primary axis and head formation during development. In this study, we use super-resolution microscopy to show that β-catenin import requires Custos as a docking protein. Custos and β-catenin form a complex in the cytoplasm and move together through the NPC into the nucleus, where they dissociate in the nucleus. Further, we provide evidence that import of β-catenin into the nucleus is a regulatory event at the NPC and define regions within the β-catenin protein required for this regulation. / Biology

In the United States, over one third of adult women are obese, and one in eight women will be diagnosed with breast cancer in their lifetime. Obesity has been shown to be a risk factor for postmenopausal breast cancer and is associated with increased aggressiveness and poor prognosis regardless of menopausal status. However, the mechanisms involved in the relationship between obesity and breast cancer are still not fully understood. Wnt signaling is often elevated in breast tumors (~60%) and is suspected to play a key role in cancer development. It has been shown that inflammatory cytokines, such as TNF-α, IL-1β, IFNγ, are potential mediators in the regulation of Wnt-signaling. We hypothesize that the low-grade inflammatory state associated with obesity is present in human mammary tissue, stimulates Wnt activity, and thereby leads to the development of breast cancer. In this project, we propose to 1) characterize the inflammatory cytokine profile, including IFN-γ, IL-1β, IL-2, IL-6, IL-8, and TNF-α, in the mammary tissue of normal weight, overweight, and obese postmenopausal women using a high performance electrochemiluminescence immunoassay; 2) determine the influences of the obesity-induced pro-inflammatory cytokines on Wnt-signaling by examining gene expression of seven Wnt-signaling target genes using real-time PCR; and 3) define the causality between TNF-α, one of the mot critical inflammatory cytokines, and Wnt signaling by measuring the gene expression of the Wnt targets in samples from normal to overweight and obese postmenopausal women treated with anti-TNF-α antibody or TNF-α recombinant protein respectively. We expect to define a novel mechanism that obesity mediates the development of postmenopausal breast via inflammation-driven Wnt signaling.

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