臺灣博碩士論文加值系統

c-Maf是一個b-Zip家族的轉錄因子，根據之前的文獻報導，c-Maf在Th2細胞中對IL-4基因調控是一個必要的存在。而最近的文獻則是報導 c-Maf與 IL-21在Th17細胞中的關係。這篇文獻發現在c-Maf KO小鼠中，IL-21的基因表現是有缺陷的，也就是說，c-Maf很有可能是調控IL-21基因表現的一個上游分子。由於c-Maf本身是一個轉錄分子，因此我們假設c-Maf會調控IL-21基因表現。為了確定c-Maf調控IL-21基因是經由直接接觸IL-21 gene locus或是透過與其它不明轉錄蛋白作用再影響到IL-21基因表現，我們利用電腦軟體在IL-21 promoter的基因序列中找尋MARE。我們在-1.3kb中總共找到兩個half MARE和兩個V-MARE。為了確定c-Maf會藉由哪一個MARE影響IL-21的表現，我們建構了具有點突變或是截短的promoter片段的pGL3質體，再利用這些質體進行Luciferase assay。我們的研究結果指出c-Maf最有與位在-192 ~ -205 kb的V-MARE作用，進而影響IL-21基因表現。除了以上的實驗，我們也去測試SUMOylation會不會讓c-Maf對IL-21基因表現造成影響。在這方面，我們的結論是：c-Maf的轉錄能力在調控IL-21基因系統中，不受到SUMOylation的影響。

c-Maf is a b-Zip transcription factor, which was found to be an essential role in regulating IL-4 expression in Th2 cells. Recent study has investigated the relationship between c-Maf and IL-21 gene expression in Th17 cells. Their study uncovered shortage of IL-21 production in c-Maf KO mice, which implied that c-Maf might be an upstream molecule of IL-21. Since c-Maf itself is a transcription factor, we hypothesized that c-Maf should regulate IL-21 gene expression. To identify whether c-Maf regulates IL-21 expression by directly or indirectly binding on IL-21 promoter, we went to investigate MARE sequence on IL-21 promoter. We found two half MARE and two V-MARE in -1.3kb upstream region. We constructed several point mutatated and truncated promoter fragments onto pGL3 Basic vector, and performed luciferase assay. Our results pointed out that the putative c-Maf binding site should be V-MARE locating -192 ~ -205kb. On the other hand, the SUMOylation effect of c-Maf on IL-21 gene expression was tested. We concluded that c-Maf transactivation ability on IL-21 gene expression was not affected by SUMOylation.

Ackowledgement…………………………………...………………….i
Abstract..………………………………………………….....................ii
摘要………………………………………………………………….. iii
Chapter I Bckground.………….…………………….................. 1
1.1 The characteristic cytokine expression of T helper cells….……... 1
1.2 Th2 cells differentiation……………………..…….…………. 2
1.3 Th17 cells differentiation…………………...………………... 3
1.4 An overview of c-Maf and its function…………………. 4
1.5 Post-translation modification of c-Maf…………………….…… 6
1.6 IL-21……………………………………………………….… 7
2. Rationale…………………………………………………….…… 8
3. Hypothesis……………………………………….... 9
Chapter II Methods……………………...………… 10
2.1 Cell Culture…………………………………………………… 10
2.2 Plasmids……………………………………………………………. 10
2.3 Transfection………………………………………………………....13
2.4 Luciferase assay………………………………….………………….13
2.5 Western Blot………………………………………………………...13
2.6 Statistics……………………………………………………………..14
Chapter III Experimental Materials................................ 15
3.1 Cell line……………………………………………………………..15
3.2 Antibodies.…………………………………………………………..15
3.3 Enzymes…………………………………………………………….15
3.4 Kits………………………………………………………………….16
3.5 Chemicals and reagents……………………………………………..16

Chapter IV Results..…………………….….…………………….19
4.1 c-Maf induce murine IL-21 gene expression………………………..19
4.2 Putative c-Maf binding sites may locate on -250bp
promoter region…………………………………….……………….20
4.3 The binding site of wt c-Maf is at V-MARE site locating
-192 ~ -205………………………………………………………….21
4.4 Sumoylation of c-Maf has no significant impact on
mIL-21 expression..………………………….……………………...22
Chapter V Discussion…...……………………………..…………24
Reference………………………………………..………………….....28
Chapter VI Experimental Figures…...…..…………… 32
Fig.1 The map of pGL3-Basic mIL-21 promoter constructs… 33
Fig.2 c-Maf is able to transactivate mIL-21 expression…................. 34
Fig.3 Two promoter deletion constructs designation……………………35
Fig.4 Slightly decreased transactivity of wt c-Maf on two
deletion mutants…………………………………………………..36
Fig.5 Three MARE mutants'' designation………………………. 37
Fig.6 Slightly decreased transactivity of wt c-Maf on two
MARE mutants..…...…………………………………….……….38
Fig.7 Parallel tendency of two wt c-Maf dosages on
four mutants………………………………………….…………….39
Fig.8 wt c-Maf failed to induce pGL3 mIL-21 promtoer
O200 expression…………..…………………………...…………..40
Fig.9 DeSumoylated environment lead to upregulated transactivity of
wt c-Maf on mIL-21 expression……………………..………..….43
Fig.10 wt c-Maf isn’t related to Sumoylation in mIL-21
expression system……………………………………………….44

Chapter VII Supplementary Data…………………………………..45
sFig.1 Slightly decreased transactivity of wt c-Maf on two
deletion mutants…………………………………………………..46
sFig.2 Slightly decreased transactivity of wt c-Maf on two
MARE mutants…………………………………………… 48
sFig.3 Parallel tendency of two wt c-Maf dosages on
four mutants………………………………………………………50
sFig.4 wt c-Maf failed to induce pGL3 mIL-21 promtoer
O200 expression………………………………………………….53

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