鋅手指蛋白74 之選殖與特性分析
The molecular cloning and characterization of zinc finger protein74 (ZNF74)
中文摘要
Notch 是個演化上具高度保留，穿過細胞膜一次的受體蛋白，在哺乳類的 Notch
受體蛋白有Notch1∼4 四種，被認為可調控基因的轉錄、細胞的增生、分化及早
期細胞命運的決定，而Notch 訊息傳遞的異常或突變會造成某些疾病的發生。當
相鄰細胞表現的ligand 與 Notch 受體蛋白的細胞外區域結合時，Notch 訊號傳遞 系統就被活化，整個細胞內區域在靠近內膜處被蛋白分解脢切斷，而形成活化 型Notch (Notch IC, 細胞內區域)。此活化型 Notch 會轉移至細胞核中，並且和一 些可與DNA 結合的轉錄因子，如：RBP-Jκ/CBF1，以及其他細胞因子結合，進
一步調控標的基因的表現。所以這些參與的因子，對於Notch 訊息傳遞路徑影響
甚巨。因此，為了進一步探討Notch 訊息傳遞路徑，本論文首要目標著重於探索
參與Notch1 下游訊息傳遞路徑仍未知的結合因子，及初步分析這種結合所產生
的生物功能。
本實驗參考葉添順老師與本實驗室先前利用GST-N1IC-ANK repeats 之融合蛋白，
與K562 細胞萃取液混合，進行 GST pull down Assay 來分析該細胞中有何蛋白
質可與GST-N1IC-ANK 融合蛋白做結合，並收集與融合蛋白結合的複合體，用
2-D 電泳及質譜儀分析此複合體的成員為何。在分析蛋白質體的數據中發現有數
個候選蛋白質包括鋅手指蛋白的存在。分析比對之結果發現ZNF74 的吻合度最
高。ZNF74 屬於 C2H2 type 的鋅手指蛋白。在其 N 端部份包含了一段稱為 Krueppel-associated box (KRAB)的區域，在 C 端部份包含 12 個 zinc finger motifs。具有 KRAB 區域的蛋白主要功能到目前為止所知均具有抑制基因表現之 功能，而zinc finger 區域則是與特定的 DNA 序列做結合。因此本實驗有興趣繼 續探討ZNF74 與 Notch 1 之訊號傳遞是否有關。在實驗第一部份我們利用專一性 引子以反轉錄聚合酶連鎖反應(RT-PCR)證實在 K562 細胞之中 ZNF74 基因是以 isoform I 存在。實驗第二部份，我們利用將 HA-ZNF74 基因植入表現 N1IC 的人 類細胞HEK293，獲得細胞萃取液後再利用 anti-HA 或 anti-N1IC 抗體來進行共 同免疫沉澱法(co-immunoprecipitation)反應，結果顯示 ZNF74 確實可以跟 N1IC 結合。為了更進一步找出ZNF74 蛋白上是利用那個區域與 N1IC 做結合，我們利 用可表現MBP 融合的不同短縮型 ZNF74 之蛋白與表現 N1IC 或 Ankyrin(Ank)的 細胞萃取液混合。結果顯示，ZNF74 的 zinc finger 區域與 Ank 的結合是必需的。
實驗最後部份，我們利用SELEX(systematic evolution of ligands by exponential enrichment )來找出 ZNF74 結合的特定核苷酸序列為何。經過軟體序列比對結果 相當複雜並無一致性。因此，根據上述的結果顯示，本實驗仍需要進一步的研究 才能夠釐清在K562 細胞中 ZNF74 蛋白參與 Notch 訊息傳遞下所扮演的生物角 色為何。
英文摘要
Notch receptors are evolutionally highly conserved and play important roles in modulating cell fate decisions throughout the development from invertebrate to vertebrate species. Four homologues of Notch receptors have been identified in human and aberrant Notch signaling is associated with a number of human diseases.
There is a consensus model of Notch signaling pathway containing multiple proteolytic steps. After ligand binding, Notch receptors are cleaved to release the intracellular domains of Notch receptors. The intracellular domains, the activated form of Notch receptors, are then translocated into nuclei and interact with other transcriptional machineries to regulate the expression of cellular genes.
In the previous data of Dr. Yeh and our lab, the proteomic study of GST-Notch1-Ank domain(GST-N1IC-Ank) pull down associated with cell lysate of erythrleukemia K562 cell, several zinc finger proteins were identified. Here, we wish to identify one of these zinc finger proteins, ZNF74, and characterize its biological function. The ZNF74 belongs to the family of Cys2-His2 type zinc finger proteins, it contains a KRAB repression domain at the amino- terminal region and 12 zinc finger motifs at carboxyl terminus. It has been showed that ZNF74 is localized to nucleus and the KRAB domain exhibits transcription repressor activity, whereas the Cys2-His2 zinc finger motifs recognized DNA sequence.
In this study, we designed to confirm the association between N1IC and ZNF74. The specific primers were synthesized and the RT-PCR result has shown that the ZNF74 transcript is splicing isoform I in the K562 cell. The mammalian expressing vector harboring HA-ZNF74 were transfected into HEK293 cells expressing N1IC, the cell lysate was prepared and immunoprecipitated by anti-HA or anti-N1-C-terminus antibody. The experiment showed that ZNF74 can associate with N1IC. For further dissecting the domains of ZNF74 associate with N1IC, we constructed different N- or C-terminal truncated ZNF74 fused to MBP protein for MBP pull down assay. The data showed that the zinc finger domain of ZNF74 is sufficient for binding with Ankyrin domain (Ank) of N1IC. The last part of this thesis, we tried to use SELEX (systematic evolution of ligands by exponential enrichment ) to find the target oligonucleotides binding sequence of ZNF74 protein. Due to the small sample size, the alignment data were complicated and difficult for interpretation. In conclusion, we still need more efforts to characterize the biological function of ZNF74 in K562 cell and to elucidate it’s relationship with Notch signaling pathway.
