楊性芳臺灣大學：分子醫學研究所林迺蕙Lin, Nai-HuiNai-HuiLin2007-11-262018-07-092007-11-262018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/51350　mcl-1屬於Bcl-2家族蛋白中具有抗細胞凋亡功能的一員，在諸多受調控的細胞生死程式中扮演重要的上游角色。組織特異基因剔除老鼠研究指出mcl-1對於早期和晚期的血球發育及維持都是不可獲缺的。Mcl-1是一個受到高度調控的基因，其表現可以被許多細胞激素或是生長因子所誘導。我們實驗室先前已經證明在Ba/F3 pro-B細胞中，IL-3主要是經由順位調控子SIE（-87）和CRE-2(-70)去誘導mcl-1基因的轉錄。為了探討這些順位調控子在生理上扮演的角色，我們製造了SIE和CRE-2突變的小鼠(Ｍcl-1mSC/mSC)。研究發現相較於野生型小鼠，突變小鼠的次級淋巴組織中CD8+　T細胞約減少了一半。同時也發現mcl-1的RNA和蛋白質表現量在突變小鼠的T細胞是比較少的，但是在B細胞中並沒有減少的現象。這些結果顯示mcl-1基因轉錄的調控在T細胞和B細胞中可能是不同的。為了更近一步的研究可能的機制，我們使用電泳速度變動分析法(EMSA)來測試T細胞和B細胞核萃取蛋白質與含有mcl-1基因啟動子片段的DNA探針反應後，比較其蛋白質核酸結合的活性有無差異。我們發現在T細胞和B細胞中在mcl-1基因啟動子-97到-65片段，都可以偵測到SIE和CRE-2 complex的形成，同時SIE和CRE-2 complex中分別具有PU.1和phsphorylated CREB。另外，我們也發現在B細胞中，mcl-1啟動子上-156位置的順位調控子(AP2-like element)上會形成B細胞較多結合的蛋白質核酸複合體(B cell enriched binding complex, BEB complex)。綜合突變小鼠的表現型分析，以及相對於T細胞，在B細胞中具有較多的BEB complex結合於mcl-1啟動子上，因此我們推測在B細胞中mcl-1基因的轉錄可能主要是受到BEB complex的調控，而在T細胞中則主要是受到SIE和CRE-2順位調控子的調控。未來尚需要更多的實驗去驗證以上的結論。Mcl-1, an anti-apoptotic member of Bcl-2 family proteins, functions at an apical step in many regulatory programs that control cell survival and death. Conditional depletion of mcl-1 in lymphoid organs suggested that it is essential for the development and maintenance of lymphocytes at both early and late stages. Mcl-1 is a highly regulated gene which can be induced by many cytokines and growth factors. Our lab has previously demonstrated that in Ba/F3 pro-B cells, IL-3 stimulation of Mcl-1 gene expression is mediated mainly through two upstream DNA motifs, which are located at positions -70 (the CRE-2 site) and -87 (the SIE site). To further investigate the physiological role of these regulatory cis-elements, the mice with mutant SIE and CRE-2 (mSC) sites were generated. A 50% decrease of peripheral CD8+T cells was found in Mcl-1mSC/ mSC mice. Mcl-1 protein and RNA levels were both decreased significantly in the T-cell lineages of Mcl-1mSC/ mSC mice but not in the B-cell lineage. These data suggested that T and B cells might have different transcription regulation of the mcl-1 gene. To determine possible mechanism responsible for this difference, EMSA was performed using radiolabeled probe containing mcl-1 promoter and nuclear extracts from lymph node T and B cells. We found that in both cell types both SIE and CRE-2 complex could be detected on the mcl-1 promoter region from –97 to –65. Besides, we confirmed that the SIE and CRE-2 complexes contained PU.1 and phosphorylated CREB, respectively. Interestingly, with the mcl-1 -203/+10 promoter DNA as a probe, a B cell enriched binding complex (BEB complex) was found to be formed on an AP2-like element at position -156 of the mcl-1 promoter. Taken together, the mutant mouse phenotype and the prominently enriched amount of the BEB complex in B cells compared with that in T cells suggest that in B cells, mcl-1 transcription is mainly regulated by the BEB complex, whereas in T cells, mcl-1 transcription is mainly controlled by both the SIE and the CRE-2 elements. Further experiments would be required to confirm this conclusion.Table of content………………1 中文摘要……………………………5 Abstract…………………6 Introduction…………7 Apoptosis……………7 Intrinsic cell death pathway…………………………………………………………………7 Extrinsic cell death pathway………………………9 Apoptosis in the development of immune system……………9 Myeloid cell leukemia-1…………………………………10 Structure organization of Mcl-1……………………………11 Mcl-1 has an anti-apoptotic function……………………11 Mcl-1 acts as an apical sensor in the apoptosis……13 Mcl-1 is not a redundant player in apoptosis control……13 Regulation of Mcl-1 expression……………………………14 Regulation of Mcl-1 protein stability………………………16 Mcl-1 interacting proteins……………………………………………16 IL-3 simulates Mcl-1 expression through the SIE and CRE-2 elements……………..17 Specific aims………………18 Material and methods………………19 Plasmids……………19 Genotyping……………………19 Primary culture of bone marrow cells and IL-3 induction…………………………….20 Western blots…………………20 RNA isolation and quantitative real time PCR……………21 Isolation of total T cells and total B cells from lymph nodes…………………22 Preparation of nuclear extracts………………………………………22 Electrophoretic mobility shift assay (EMSA)……………23 Flow cytometric analysis………………………………………………24 Results………………………………26 Generation of Mcl-1mSC/mSC mice………………………26 The numbers of peripheral CD8+ single positive T cells are decreased in the Mcl-1mSC/mSC mice…………………………………………27 Stimulation of Mcl-1 expression of the Mcl-1mSC/mSC mice is attenuated in the bone marrow cells……………………………………29 Mcl-1 protein is specifically decreased in the T cell linage of lymphoid organs from Mcl-1mSC/mSC mice……………………………………………29 Mcl-1 RNA was significantly decreased in lymph node T cells, but not B cells of the Mcl-1mSC/mSC mice…………………………………………………30 Mcl-1 protein levels were not significantly altered in the non-lymphoid organs of the Mcl-1mSC/mSC mice………………………………………………………31 Both T and B cells have the SIE and CRE-2 specific binding complex.…………31 PU.1 and phosphorylated CREB are involved in the SIE and CRE-2 specific binding complexes, respectively……………………………………………………32 B cell nuclear extracts have another specific binding complex recognizing sequence within the mcl-1 (-203/+10) promoter fragment………………………………33 BEB complex recognizes a sequence within the upstream half of the mcl-1 (-203/+10) promoter……………………34 DNA motif containing an AP2-like element is the binding site of the BEB complex in the B cells………………………35 BCL1 and P3X cells have the AP2 specific BEB complex………………………35 Discussion…………………………37 A 50% reduction of peripheral CD8+ T cells in Mcl-1mSC/mSC mice is mainly attributed by mutation in the CRE-2 element……………………………………………37 Impaired survival leads to reduced numbers of CD8+ T cells in the Mcl-1mSC/mSC mice…………………………………………38 CD8+ T cell is more sensitive than other T cell lineages to reduced expression of Mcl-1………………………………39 The BEB complex may play a major role in transcription regulation of the mcl-1 gene in B cells…………………………………………………40 Figure……………42 FIGURE 1. General model of signaling pathways that mediate apoptosis………42 FIGURE 2. The mammalian BCL-2 family members…………………………43 FIGURE 3. mcl-1 is an immediate-early gene activated by the interleukin 3 (IL-3) signaling pathways……………………………………………………44 FIGURE 4. Targeted mutation of the mcl-1 gene promoter………………………45 FIGURE 5. Genotyping and Mendelian ratio…………………………………46 FIGURE 6. Flow cytometric analysis of hematopoietic cell compositions of Mcl-1wt/wt and Mcl-1mSC/mSC mice…………………………………………47 FIGURE 7. Early T cell development is normal in the thymus of Mcl-1mSC/mSC mice………………………48 FIGURE 8. Myeloid cell development is slightly attenuated in the bone marrow of Mcl-1mSC/mSC mice………………………………………49 FIGURE 9. CD8+ SP T cell deficiency in the spleen of Mcl-1mSC/mSC mice………50 FIGURE 10. CD8+ T cell deficiency in the lymph node of Mcl-1mSC/mSC mice…51 FIGURE 11. IL-3 induction of Mcl-1 protein in the bone marrow cells is attenuated in Mcl-1mSC/mSC mice………………………………………………52 FIGURE 12. The IL-3 induction of Mcl-1 RNA in the bone marrow cells of Mcl-1mSC/mSC mice is less than that of Mcl-1wt/wt mice…………………………53 FIGURE 13. Mcl-1 expression is attenuated in the T-cell lineage of Mcl-1mSC/mSC mice………………………54 FIGURE 14. Mcl-1 mRNA is significantly reduced in lymph node T cells, but not B cells of Mcl-1mSC/mSC mice……………………………………………………………55 FIGURE 15. No consistent difference in Mcl-1 levels in the non-lymphoid organs of Mcl-1wt/wt and Mcl-1mSC/mSC mice………………………………………………………56 FIGURE 16. Complex formed on the SC fragment………………………………57 FIGURE 17. The SC promoter region binds proteins complexes contained PU.1 and/or phosphorylated CREB………………………………………………58 FIGURE 18. Schematic representation of mcl-1 -203/+10 WT and mutant promoter…………………………59 FIGURE 19. The mcl-1 promoter contains an abundant protein complex (BEB) in the lymph node B cells……………………………………………………60 FIGURE 20. The BEB complex does not contain PU.1 or phosphorylated CREB...61 FIGURE 21. The BEB complex is formed on the mcl-1 promoter between -203 and -87 region…………………………………62 FIGURE 22. Identification of the nuclear protein binding site of the BEB complex……………………………………63 FIGURE 23. The BEB complex specifically forms on the AP2 element (at position -156) within the mcl-1 promoter………………………………………………………64 FIGURE 24. BCL1, A20 and P3X cells have a BEB-like complex formed on the mcl-1 promoter........65 FIGURE 25. The BEB-like complex formed on the mcl-1 promoter in the BCL1 mature B cells is very similar to that formed in the CD19+ primary B cells……66 FIGURE 26. The BEB-like complex is also formed in the P3X plasma B cells……67 Reference………681725658 bytesapplication/pdfen-USmcl-1轉錄T淋巴球細胞B淋巴球細胞transcriptionT lymphocytesB lymphocytesotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51350/1/ntu-95-R93448005-1.pdf