臺灣博碩士論文加值系統

成熟的紅血球是從早期的多功能造血幹細胞(pluripotent hematopoietic stem cells)經由一連串複雜的分化過程而來。Erythroid Krüppel-like Factor (EKLF) 在紅血球分化的過程中是不可或缺的一個轉錄因子，尤其又以調控後期分化更為重要。EKLF 已在小鼠及人類中被證實對於活化成人時期乙型血紅蛋白(β globin)是必須的，在轉錄活化的調控機制中顯示EKLF會和CBP/p300 及SWI/SNF 蛋白質複合體作用而結合到乙型血紅蛋白的啟動子區域進而影響到染色質重塑(chromatin remodeling)。在EKLF基因剔除小鼠中觀察到紅血球的分化及成熟過程受到影響。為了研究EKLF在紅血球分化過程中的調控機制，我們利用DNA 微陣列(Microarray)及染色質免疫沉澱微陣列(ChIP on chip)的方法確定EKLF直接調控的下游基因並探討它們在紅血球分化的過程中所執行的功能為何。首先利用半定量反轉錄聚合酶鏈式反應(semi-quantitative RT-PCR)驗證在DNA微陣列中的基因其表現量在EKLF基因剔除小鼠中下降者定義為受EKLF活化的基因(EKLF up-regulated genes)，而基因其表現量上升者定義為受EKLF抑制的基因(EKLF down-regulated genes)。我們驗證DNA微陣列的準確度超過百分之八十。在功能缺失性實驗(loss-of-function study)中缺乏EKLF及處理DMSO促使細胞分化的情況下，受到活化的基因表現量會下降，顯示EKLF對於基因的調控是必須的且位在上游。除此之外我們也觀察這些受到活化或抑制的基因在MEL細胞株分化過程中的表現量情況。我們目前的實驗證實EKLF不只能扮演轉錄活化因子也能扮演轉錄抑制因子。另一方面，我們也對於EKLF在進核後會被SUMO蛋白質修飾化其所扮演的功能感興趣，因此利用EKLF SUMO修飾化位置突變的小鼠來研究SUMO蛋白質修飾化對EKLF的功能影響。在EKLF SUMO位置突變的小鼠中觀察到網狀紅血球(reticulocyte)的數目上升且紅血球的數目下降，我們假定在網狀紅血球分化為成熟紅血球的過程中出現問題。為了找出EKLF SUMO修飾化的功能，我們利用DNA 微陣列(Microarray)及染色質免疫沉澱微陣列(ChIP on chip)的方法找出EKLF SUMO修飾化後可能調控的下游基因，進而發現EKLF可能是藉由調控一個基因為Atg7(Autophagy related 7)其已知參與在網狀紅血球分化為成熟紅血球過程中的粒線體清除(Mitochondria clearance)步驟中。而Atg7在EKLF SUMO位置突變的小鼠中表現量是下降的。因此我們推論EKLF SUMO修飾化後會調控Atg7進而幫助粒線體清除使網狀紅血球分化為成熟紅血球。

Erythropoiesis is a multi-step process for production of red blood cells from pluripotent hematopoietic stem cells (HSCs). Erythroid Krüppel-like Factor (EKLF) is a critical transcription factor that functions in erythropoiesis, in particular, the erythroid terminal differentiation. It has been demonstrated that EKLF is required for the activation of adult β globin gene in mouse and human. The transactivation model shows that it participates in the remodeling of the chromatin through interactions with CBP/p300 and the SWI/SNF complex at the β globin gene promoter. EKLF knockout mice showed the failure of erythroid terminal differentiation. In order to investigate the regulation mechanism of EKLF during erythroid differentiation, we performed the ChIP on chip assay and expression profiling microarray analysis to identify the EKLF downstream target genes functioning in erythroid terminal differentiation. First, we validates the expression of differential genes in microarray data by semi-quantitative RT-PCR, the expression of targets will decrease in EKLF knockout mice identified as EKLF up-regulated genes, and the expression of targets will increase identified as EKLF down-regulated genes. We validated the accuracy of the differential expression profiling microarray was over 80%. The loss-of-function study shows that knockdown of EKLF under DMSO treatment that promote cell differentiation reduced the targeted genes mRNA expression level. In addition, we also observed their expression level during MEL differentiation. Our current data conclude that EKLF occupancy in restricted promoter, and acts as not only a transcriptional activator but a transcriptional repressor. On the other hand, we also interested in the function of sumoylation of EKLF during differentiation. We performed the EKLF K74R knock-in mice to investigate the function of sumoylation of EKLF. We observed the increase of reticulocyte number and the reduction of RBC number in our knock-in mice, indicating that there were some defects during reticulocyte to RBC transition. In order to investigate the function of sumoylated EKLF, we combined the ChIP-chip and expression microarray analysis to find the sumoylated EKLF downstream target genes. We found a downstream gene named Atg7 (Autophagy related 7) which has been demonstrated it involved in mitochondrial clearance during reticulocyte to RBC transition. The expression of Atg7 showed the down regulation in EKLF K74R knock-in mice. We speculated sumoylated EKLF might regulate Atg7 to promote mitochondria clearance during reticulocyte to RBC transition.

目錄
1.論文審定同意書.............................................i
2.誌謝....................................................ii
3.中文摘要................................................iii
4.英文摘要..................................................v
5.Introduction.............................................1
6.Materials and Methods....................................5
7.Results..................................................8
7.1 EKLF knockout E14.5 embryo is anaemic and fetal liver is paler than wild-type. .....................................8
7.2 Semi-quantitative RT-PCR validation of expression profiling microarray.......................................8
7.3 Analysis of EKLF binding signal in ChIP-chip data......9
7.4 Gain-of-function study failed to demonstrate the targeted genes were activated by EKLF......................9
7.5 Loss-of-function study revealed that EKLF might be upstream of the targeted genes during erythroid differentiation...........................................10
7.6 Analysis of the targeted genes mRNA expression level during MEL differentiation revealed that the targeted genes may function in erythroid terminal differentiation........11
7.7 Analysis of EKLF K74R knock-in mice expression profiling microarray and ChIP-chip data to find out the associated functional network........................................12
7.8 Analysis of the targeted genes mRNA expression level during MEL differentiation................................13
7.9 Analysis of EKLF binding signal in ChIP-chip data of EKLF K74R knock-in mice...................................13
7.10 Construction of Atg7 promoter into pGL3-basic luciferase reporter plasmid to perform the reporter assay.14
8. Discussions............................................15
8.1 Difference of cell population between wild-type and EKLF knockout mice E14.5 fetal liver might affect the accuracy of expression profiling microarray...........................15
8.2 Limitation of validation of expression profiling microarray by semi-quantitative RT-PCR....................15
8.3 Gain-of-function study failed to activate the targeted genes expression..........................................16
8.4 Loss-of-function study showed the regulation of EKLF and targeted genes............................................16
8.5 The targeted genes play some roles in stage-specific regulation during erythroid differentiation...............17
8.6 To investigate the function of sumoylation of EKLF in EKLF K74R knock-in mice model.............................17
8.7 The Difference of cell populations between wil- type and EKLF K74R knock-in E14.5 fetal liver......................18
9. References.............................................19
10. Figures...............................................24
10.1 Figure 1. Phenotypes and genome typing result of wild type (+/+) and EKLF knockout (-/-) E14.5 fetuses.........24
10.2 Figure 2. Semi-quantitative RT-PCR validation of expression profiling microarray database..................25
10.3 Figure 3. Gain-of-function study shows EKLF overexpression failed to activate downstream targeted genes. ..........................................................26
10.4 Figure 4. Loss-of-function study shows inducible knockdown EKLF under DMSO treatment reduced the expression level of downstream targeted genes........................27
10.5 Figure 5. Quantitative data of loss-of-function study in figure 4...............................................29
10.6 Figure 6. Expression pattern of EKLF up-regulated genes during erythroid differentiation..........................30
10.7 Figure 7. Quantitative data of expression pattern in EKLF up-regulated gens during erythroid differentiation in figure 6..................................................32
10.8 Figure 8. Expression pattern of down-regulated genes during erythroid differentiation..........................33
10.9 Figure 9. Quantitative data of expression pattern in EKLF down-regulated gens during erythroid differentiation in figure 8..................................................35
10.10 Figure 10. Associated functional network of EKLF K74R knock-in mice.............................................36
10.11 Figure 11. Validation of expression profiling microarray (WT compared to Kin and KO) by semi-quantitative RT-PCR....................................................37
10.12 Figure 12. Expression pattern of EKLF K74R knock-in related genes during erythroid differentiation............38
10.13 Figure 13. Quantitative data of expression pattern in EKLF K74R knock-in related gens during erythroid differentiation in figure 8...............................39
10.14 Figure 14. Genome location of EKLF binding site in Atg7 promoter.............................................40
10.15 Figure 15. Construction of Atg7 promoter into pGL3-basic luciferase reporter plasmid.........................41
11. Tables................................................42
11.1 Table 1. EKLF up-regulated genes in expression profiling microarray......................................42
11.2 Table 2. EKLF down-regulated genes in expression profiling microarray......................................43
11.3 Table 3. The binding location of EKLF-up regulated genes in the expression profiling microarray..............44
11.4 Table 4. The binding location of EKLF-down regulated genes in the expression profiling microarray..............44
11.5 Table 5. EKLF K74R knock-in regulated genes in expression profiling microarray...........................45
11.6 Table 6. The binding location of EKLF K74R knock-in regulated genes in expression profiling microarray........45
11.7 Supplementary Table 1. Primer sequences for semi-quantitative RT-PCR (EKLF up-regulated gene)..............46
11.8 Supplementary Table 2. Primer sequences for semi-quantitative RT-PCR (EKLF down-regulated gene.............46
11.9 Supplementary Table 3. Primer sequences for semi-quantitative RT-PCR.......................................47
11.10 Supplementary Table 4. Primer sequences for semi-quantitative RT-PCR (EKLF K74R regulated gene)............47

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