臺灣博碩士論文加值系統

時間以及空間上的調控，對於生物體的發育非常重要。雖然在生物體發育的過程中，空間上如何被調控的機制的研究已經相當多，但是對於時間上如何被準確調控卻知道的非常少。在線蟲當中，有一群異時調控的基因，能夠去控制特定的發育時間點。在這些異時調控的基因當中，dre-1、lin-29 以及daf-12 共同調控線蟲遠端細胞移動的時間。實驗室先前的研究當中發現，這三個異時調控基因，共同去調控了一個轉錄因子BLMP-1，進而去調控遠端細胞的移動。其中lin-29 以及daf-12，主要是藉由抑制了BLMP-1 的轉錄去調控調控遠端細胞。然而，另一個異時調控基因dre-1 是如何去調控BLMP-1 尚未清楚。因此在本篇研究當中，主要是去研究dre-1 是藉由何種機制來調控BLMP-1 的表現。已知DRE-1 是一種F-box protein，是SCF E3 ligase complex 的組成之一，因此BLMP-1 可能藉由被DRE-1 加上泛素而降解。利用免疫共沈澱的方法，發現BLMP-1 能夠和DRE-1 相互作用。接下來，利用HEK 293T 細胞表現這兩種蛋白之後，發現BLMP-1 以及DRE-1 都是藉由進行蛋白酶體降解，而且BLMP-1的降解是經由DRE-1 來達成的。因為在人類中有一個轉錄因子PRDI-BF1 和BLMP-1 非常相似，而且也參與在許多發育以及腫瘤的生成。另外，DRE-1 在
人類的蛋白當中也有一個非常相似的蛋白FBXO11。因為先前已經發現兩個線蟲的蛋白有交互作用，所以在免疫共沈澱的結果，PRDI-BF1 也和FBXO11 有交互作用，顯示這兩個蛋白之間調控的關係在演化上是有保守性的。在本篇研究當中，我們提供了一個參與在發育過程中新的調控關係，此調控關係從線蟲到人類都是有保守性的。

The spatiotemporal control of gene expression is crucial for the development of multicellular organisms. Although the studies in spatial patterning are extensive, temporal regulation of development is poorly understood. In C. elegans, several heterochronic genes, which function in the timing of specific developmental stages, have been identified and characterized. Among them, dre-1, lin-29 and daf-12 act redundantly to control the temporal migration pattern of somatic gonadal cells, distal tip cells (DTC). Our laboratory found that BLMP-1, a zinc finger transcription repressor orthologous to mammalian Blimp-1 (B lymphocyte-induced maturation protein 1), acts downstream of these heterochronic genes to regulate DTC migration path. Blmp-1 is transcriptionally down-regulated by lin-29 and daf-12. However, the regulation of BLMP-1 by DRE-1 is still unclear. My thesis project focused on determining whether BLMP-1 is also down-regulated by DRE-1. Because dre-1 encodes an F-box protein, which functions in SCF E3 ligase complexes, I hypothesize that DRE-1 regulates BLMP-1 protein stability through ubiquitin-mediated proteolysis. Using co-immunoprecipitation assays, I found that DRE-1 and BLMP-1 physically interact with each other. I next assessed the stability of BLMP-1 and DRE-1 by transiently expressing either proteins in HEK 293T cells and found that the turnover of both DRE-1 and BLMP-1 are dependent on proteasome. When co-expressed with DRE-1 in HEK293T cells, the stability of BLMP-1 decreases, indicating the
degradation of BLMP-1 is dependent on DRE-1. Because the mammalian homolog of BLMP-1, PRDI-BF1, has been shown to participate in various cellular processes, I next tested whether PRDI-BF1 is also regulated through distinct F-box proteinmediated degradation. I found that PRDI-BF1 co-immunoprecipitated with FBXO11, the mammalian homolog of DRE-1. These results support an evolutionarily conserved DRE-1/FBXO11–dependent degradation of BLMP-1/PRDI-BF1 in both mammals and nematodes. Based on the discovery in C. elegans, we provide an evolutionary conserved regulation that may involve in organism development.

口試委員審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
Contents vi
Chapter I Introduction 1
Chapter II Materials and Methods 6
1. Materials 6
1.1. Cell culture 6
1.2. Antibodies 6
2. Methods 6
2.1. Nematode strains 6
2.2. RNA interference 7
2.3. Cell culture 7
2.4. Constructs 7
2.5. Cell lysate preparation 8
2.6. Co-Immonoprecipitation 9
2.7. Western blotting 9
Chapter III Results 11
SCF complex regulates DTC dorsal turn. 11
BLMP-1 interacts with DRE-1. 12
DRE-1 mediates the degradation of BLMP-1 through proteasome-dependent pathway. 13
The degradation of DRE-1 is proteasome-dependent 13
BLMP-1 PR domain deletion mutant destabilizes DRE-1 but is not essential for DRE-1 binding. 14
PRDI-BF1 interacts with FBXO11 that’s evolution conserved. 15
The regulation of FBXO11 is conserved to DRE-1 in human and C. elegans. 16
Chapter IV Discussion 17
Timely removal of BLMP-1 is crucial for DTC dorsal migration 17
Anterior and posterior DTCs response differentially to the SCF complex during DTC dorsal turn. 18
SCF complex promotes DTC dorsal turn not only depends on promoting BLMP-1 degradation. 19
The down-regulation of BLMP-1 in HEK 293T cell indicates that the protein structure is conserved to PRDI-BF1. 19
F-box proteins are actively turned-over in cells. 20
The potential mechanism of BLMP-1 recognizes by DRE-1. 21
The interaction between BLMP-1 and DRE-1 in C. elegans is conserved to PRDI-BF1 and FBXO11 in humans 22
References 23
Figures 26
Figure 1. Schematic pictures show protein structures, gonad morphogenesis and SCF complex. 27
Figure 2. DRE-1 interacts with BLMP-1. 28
Figure 3. BLMP-1 also interacts with DRE-1. 29
Figure 4. The degradation of BLMP-1 is proteasome-mediated pathway that is dependent on DRE-1. 30
Figure 5. DRE-1 is degraded through proteasome dependent pathway. 31
Fig 6. The interaction between DRE-1 and BLMP-1 PR domain decreases, resulting in the DRE-1 instability. 32
Figure 7. PRDI-BF1 interacts with FBXO11. 33
Figure 8. FBXO11, a homolog of DRE-1 in human, is also degraded through proteasome dependent pathway. 35
Tables 36

Albert, M., Kim, J., and Birge, R. (2000). alphavbeta5 integrin recruits the CrkII-Dock180-rac1 complex for phagocytosis of apoptotic cells. Nature cell biology 2, 899-905.
Asakura, T., Ogura, K.-i., and Goshima, Y. (2007). UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons. Developmental biology 304, 800-810.
Blelloch, R., Anna-Arriola, S., Gao, D., Li, Y., Hodgkin, J., and Kimble, J. (1999). The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans. Developmental biology 216, 382-393.
Blelloch, R., and Kimble, J. (1999). Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans. Nature 399, 586-590.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Chan, S., Zheng, H., Su, M., Wilk, R., Killeen, M., Hedgecock, E., and Culotti, J. (1996). UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Cell 87, 187-195.
Chen, Y., Yang, Z., Meng, M., Zhao, Y., Dong, N., Yan, H., Liu, L., Ding, M., Peng, H., and Shao, F. (2009). Cullin mediates degradation of RhoA through evolutionarily conserved BTB adaptors to control actin cytoskeleton structure and cell movement. Molecular cell 35, 841-855.
Chiorazzi, M., Rui, L., Yang, Y., Ceribelli, M., Tishbi, N., Maurer, C., Ranuncolo, S., Zhao, H., Xu, W., Chan, W.-C., et al. (2013). Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma. Proceedings of the National Academy of Sciences of the United States of America 110, 3943-3948.
Deppe, U., Schierenberg, E., Cole, T., Krieg, C., Schmitt, D., Yoder, B., and von Ehrenstein, G. (1978). Cell lineages of the embryo of the nematode Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America 75, 376-380.
Duan, S., Cermak, L., Pagan, J., Rossi, M., Martinengo, C., di Celle, P., Chapuy, B., Shipp, M., Chiarle, R., and Pagano, M. (2012). FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Nature 481, 90-93.
Fielenbach, N., Guardavaccaro, D., Neubert, K., Chan, T., Li, D., Feng, Q., Hutter, H., Pagano, M., and Antebi, A. (2007). DRE-1: an evolutionarily conserved F box protein that regulates C. elegans developmental age. Developmental cell 12, 443-455.
Ho, M., Ou, C., Chan, Y.R., Chien, C.T., and Pi, H. (2008). The utility F-box for protein destruction. Cellular and molecular life sciences : CMLS 65, 1977-2000.
Horvitz, H., and Sternberg, P. (1982). Nematode postembryonic cell lineages. Journal of nematology 14, 240-248.
Jin, J., Cardozo, T., Lovering, R., Elledge, S., Pagano, M., and Harper, J. (2004). Systematic analysis and nomenclature of mammalian F-box proteins. Genes & development 18, 2573-2580.
Kim, H.-S., Murakami, R., Quintin, S., Mori, M., Ohkura, K., Tamai, K., Labouesse, M., Sakamoto, H., and Nishiwaki, K. (2011). VAB-10 spectraplakin acts in cell and nuclear migration in Caenorhabditis elegans. Development (Cambridge, England) 138, 4013-4023.
Kovacevic, I., Ho, R., and Cram, E. (2012). CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans. Mechanisms of development 128, 548-559.
Lin, K.-I., Angelin-Duclos, C., Kuo, T., and Calame, K. (2002). Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Molecular and cellular biology 22, 4771-4780.
Meighan, C., and Schwarzbauer, J. (2007). Control of C. elegans hermaphrodite gonad size and shape by vab-3/Pax6-mediated regulation of integrin receptors. Genes & development 21, 1615-1620.
Merz, D., Zheng, H., Killeen, M., Krizus, A., and Culotti, J. (2001). Multiple signaling mechanisms of the UNC-6/netrin receptors UNC-5 and UNC-40/DCC in vivo. Genetics 158, 1071-1080.
Min, S.-H., Lau, A., Lee, T., Inuzuka, H., Wei, S., Huang, P., Shaik, S., Lee, D., Finn, G., Balastik, M., et al. (2012). Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase. Molecular cell 46, 771-783.
Nishiwaki, K., Hisamoto, N., and Matsumoto, K. (2000). A metalloprotease disintegrin that controls cell migration in Caenorhabditis elegans. Science (New York, NY) 288, 2205-2208.
Ogura, K.-i., Asakura, T., and Goshima, Y. (2012). Localization mechanisms of the axon guidance molecule UNC-6/Netrin and its receptors, UNC-5 and UNC-40, in Caenorhabditis elegans. Development, growth & differentiation 54, 390-397.
Reddien, P., and Horvitz, H. (2000). CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nature cell biology 2, 131-136.
Rossi, M., Duan, S., Jeong, Y.-T., Horn, M., Saraf, A., Florens, L., Washburn, M., Antebi, A., and Pagano, M. (2013). Regulation of the CRL4(Cdt2) ubiquitin ligase and cell-cycle exit by the SCF(Fbxo11) ubiquitin ligase. Molecular cell 49, 1159-1166.
Shaffer, A., Lin, K., Kuo, T., Yu, X., Hurt, E., Rosenwald, A., Giltnane, J., Yang, L., Zhao, H., Calame, K., et al. (2002). Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 17, 51-62.
Shaffer, A., Yu, X., He, Y., Boldrick, J., Chan, E., and Staudt, L. (2000). BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity 13, 199-212.
Su, M., Merz, D., Killeen, M., Zhou, Y., Zheng, H., Kramer, J., Hedgecock, E., and Culotti, J. (2000). Regulation of the UNC-5 netrin receptor initiates the first reorientation of migrating distal tip cells in Caenorhabditis elegans. Development (Cambridge, England) 127, 585-594.
Sulston, J., Schierenberg, E., White, J., and Thomson, J. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental biology 100, 64-119.
Wadsworth, W., Bhatt, H., and Hedgecock, E. (1996). Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 16, 35-46.
Xu, X., Rongali, S., Miles, J., Lee, K., and Lee, M. (2006). pat-4/ILK and unc-112/Mig-2 are required for gonad function in Caenorhabditis elegans. Experimental cell research 312, 1475-1483.
Zhang, Q., Chen, Q., Lu, X., Zhou, Z., Zhang, H., Lin, H.Y., Duan, E., Zhu, C., Tan, Y., and Wang, H. (2013). CUL1 promotes trophoblast cell invasion at the maternal-fetal interface. Cell death & disease 4.