在K562細胞中Gfi-1B基因表現的調控機制

Abstract

中文摘要： Gfi-1B（growth factor independence 1B）為細胞原致癌基因，其所譯碼的蛋白質之胺基端有SNAG repressor domain，

Abstract Gfi-1B（growth factor independence-1B）is a proto-oncogene that encodes a transcriptional repressor with an N-terminal SNAG repressor domain and a C-terminal zinc finger domain. Expression of Gfi-1B is restricted to erythroid lineage cells and is essential for erythropoiesis. As Gfi-1B is highly expressed in the erythroid lineage chronic myelocytic leukemia (CML) line K562, this study aims to understand how the expression of Gfi-1B is regulated in K562 cells. Toward understanding the transcriptional control of the human Gfi-1B gene, I first defined its transcription start site. By using oligo-capping method, its first non-coding exon of Gfi-1B gene was found to be approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, I have demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, this GATA-1-mediated trans-activation not only is dependent on its binding to the promoter, but also requires transcription factor NF-Y binding to the CCAAT site. Thus, functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter. The expression of many eukaryotic transcription factors has been shown to be autoregulated positively and negatively. To further investigating the effect of Gfi-1B on its own promoter. By ectopic expression of Gfi-1B in K562 cells, I have demonstrated that the transcription of Gfi-1B is negatively regulated by its own gene product. GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter, and Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that expression of Gfi-1B is negatively autoregulated through GATA-1, but also suggest that Gfi-1B can modulate transcription in erythroid-type cells without its direct interaction with the Gfi-1 site of the target genes. Here, I propose that this negative autoregulatory feedback loop provides a mean to restrict the expression level of Gfi-1B, thus limiting its inhibitory effect on GATA-1-mediated transcription necessary for erythroid differentiation. In addition to GATA-1 mediated transcription, I also found that Gfi-1B expression in K562 cells is controlled by post-transcriptional and post-translational regulation. RNA transcript and polypeptide of Gfi-1B were found to be destabilized in response to phorbol ester (PMA) treatment in K562 cells. Degradation of Gfi-1B protein is through the ubiquitin-proteasome-mediated pathway. During PMA treatment, Gfi-1B became phosphorylated in its PEST containing region. However, the phosphorylation on PEST sequence is not a requisite signal for Gfi-1B degradation during PMA treatment in K562 cells. In summary, the results obtained from this study indicated that multiple layers of regulation interplay to confer a tight regulation on Gfi-1B expression in K562 cells.