| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 94 === Mcl-1, an anti-apoptotic member of Bcl-2 family proteins, functions at an apical step in many regulatory programs that control cell survival and death. Conditional depletion of mcl-1 in lymphoid organs suggested that it is essential for the development and maintenance of lymphocytes at both early and late stages. Mcl-1 is a highly regulated gene which can be induced by many cytokines and growth factors. Our lab has previously demonstrated that in Ba/F3 pro-B cells, IL-3 stimulation of Mcl-1 gene expression is mediated mainly through two upstream DNA motifs, which are located at positions -70 (the CRE-2 site) and -87 (the SIE site). To further investigate the physiological role of these regulatory cis-elements, the mice with mutant SIE and CRE-2 (mSC) sites were generated. A 50% decrease of peripheral CD8+T cells was found in Mcl-1mSC/ mSC mice. Mcl-1 protein and RNA levels were both decreased significantly in the T-cell lineages of Mcl-1mSC/ mSC mice but not in the B-cell lineage. These data suggested that T and B cells might have different transcription regulation of the mcl-1 gene. To determine possible mechanism responsible for this difference, EMSA was performed using radiolabeled probe containing mcl-1 promoter and nuclear extracts from lymph node T and B cells. We found that in both cell types both SIE and CRE-2 complex could be detected on the mcl-1 promoter region from –97 to –65. Besides, we confirmed that the SIE and CRE-2 complexes contained PU.1 and phosphorylated CREB, respectively. Interestingly, with the mcl-1 -203/+10 promoter DNA as a probe, a B cell enriched binding complex (BEB complex) was found to be formed on an AP2-like element at position -156 of the mcl-1 promoter. Taken together, the mutant mouse phenotype and the prominently enriched amount of the BEB complex in B cells compared with that in T cells suggest that in B cells, mcl-1 transcription is mainly regulated by the BEB complex, whereas in T cells, mcl-1 transcription is mainly controlled by both the SIE and the CRE-2 elements. Further experiments would be required to confirm this conclusion.
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