Gfi-1B於造血細胞內之轉錄抑制功能及其核酸穩定性之調節機制

Gfi-1B屬於Gfi家族之轉錄抑制因子(transcriptional repressor），是紅血球新生作用(erythropoiesis)過程的必要分子。在紅血球前驅細胞 (erythroid progenitor cells)中，表現大量Gfi-1B，可以造成這些細胞在7至10天後大量凋亡(apoptosis) 的現象。同時，抗凋亡蛋白質Bcl-xL的表現量也明顯的減少。因此，本研究首要目的，在於瞭解Gfi-1B在Bcl-xL之轉錄調節之角色。由於GATA-1亦是一個紅血球生成之必要轉錄因子，本實驗室過去的研究發現Gfi-1B可和GATA-1結合。因此，在本論文研究中，利用染色質沈澱分析法(ChIP)得知，在紅血球前驅細胞分化過程中，GATA-1始終都與Bcl-x的啟動子(promoter)結合，Gfi-1B只有分化前期才會與Bcl-x的啟動子結合。GATA-1可結合在Bcl-x啟動子上的GATT位置，誘發Bcl-xL基因表現，過度表現Gfi-1B則可以抑制GATA-1對Bcl-xL的活化。Gfi-1B是透過與GATA-1的結合而被帶領到Bcl-x啟動子上，並進行轉錄抑制。此外，在K562細胞加入Bcr-Abl激酶抑制劑Imatinib可以導致Gfi-1B的表現量上升。此時，Gfi-1B也可與GATA-1結合並抑制Bcl-xL的基因表現。利用干擾性核醣核酸(RNA interference)降低Gfi-1B的表現可以減少Imatinib所引發的細胞凋亡。另一方面，在K562細胞中過度表現Gfi-1B則會使細胞對砷化物(arsenic)所引發的死亡更為敏感。以上的實驗結果，闡釋了Gfi-1B在紅血球細胞內協同GATA-1參與誘導細胞凋亡之角色。此外，本論文亦研究Gfi-1B的後轉錄調控機制(post-transcriptional control)，並發現Gfi-1B的訊息核醣核酸(mRNA)半衰期短，該Gfi-1B mRNA具一可造成該核醣核酸不穩定的片段。同時，其 mRNA上之負面調節因子是有賴於轉譯作用以利RNA分解之進行。

關鍵字

轉錄抑制；核酸穩定性

並列摘要

Gfi-1B (growth factor independence-1B) gene is a Gfi-family transcriptional repressor, whose expression plays an essential role in erythropoiesis. Early erythroid progenitor cells overexpressing Gfi-1Bexhibit massive apoptosis after 7-10 days of culture with a significant reduction of Bcl-xL expression. Therefore, the aim of this study is to uncover the role of Gfi-1B in the regulation of Bcl-xL transcription GATA-1 is also an essential transcriptional factor in erythropoiesis. Our laboratory previously showed that Gfi-1B interacts with GATA-1. Using ChIP assays, I provide evidence that GATA-1 binds to the Bcl-x promoter constantly in the entire induction period, while Gfi-1B is transiently associated with the promoter in the early phase. GATA-1 binds to the noncanonical GATT motif of the Bcl-x promoter for trans-activation, and that enforced expression of Gfi-1B represses the Bcl-x promoter in a GATA-1-dependent manner. Gfi-1B expressed at increased levels is recruited to the Bcl-x promoter through its association with GATA-1, suppressing Bcl-xL transcription. Furthermore, I showed that imatonob, Bcr-Abl kinase inhibitor, causes up-regulation of Gfi-1B in K562 cells, where it also cooperated with GATA-1 to repress Bcl-xL transcription. Gfi-1B knockdown by RNA interference diminished imatinib-induced apoptosis, while the overexpression of Gfi-1B sensitized K562 cells to arsenic-induced death. These findings illuminate the role of Gfi-1B in GATA-1-mediated transcription in the survival aspect of erythroid cells. In addition, the post-transcriptional control of the Gfi-1B gene is studied. It was found that Gfi-1B mRNA is a short half-life transcript containing a destabilizing element within the coding region of the Gfi-1B mRNA. I further defined the region of the Gfi-1B coding region determinant (CRD) and demonstrated that this CRD confers cis-directed destabilization coupled with translation.

並列關鍵字

transcriptional repression ； RNA stability

參考文獻

1. Anderson, J. R., D. Mukherjee, K. Muthukumaraswamy, K. C. Moraes, C. J. Wilusz, and J. Wilusz. 2006. Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome. Rna 12:1810-6.

2. Bachurski, C. J., N. G. Theodorakis, R. M. Coulson, and D. W. Cleveland. 1994. An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs. Mol Cell Biol 14:4076-86.

3. Baetz, K., and B. Andrews. 1999. Regulation of cell cycle transcription factor Swi4 through auto-inhibition of DNA binding. Mol Cell Biol 19:6729-41.

6. Bernstein, P., S. W. Peltz, and J. Ross. 1989. The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol Cell Biol 9:659-70.

7. Bernstein, P. L., D. J. Herrick, R. D. Prokipcak, and J. Ross. 1992. Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant. Genes Dev 6:642-54.

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Gfi-1B於造血細胞內之轉錄抑制功能及其核酸穩定性之調節機制

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