探討SIE和CRE-2順位調控子在T細胞和B細胞中對於Mcl-1轉錄調控所扮演的角色

Abstract

mcl-1屬於Bcl-2家族蛋白中具有抗細胞凋亡功能的一員，在諸多受調控的細胞生死程式中扮演重要的上游角色。組織特異基因剔除老鼠研究指出mcl-1對於早期和晚期的血球發育及維持都是不可獲缺的。Mcl-1是一個受到高度調控的基因，其表現可以被許多細胞激素或是生長因子所誘導。我們實驗室先前已經證明在Ba/F3 pro-B細胞中，IL-3主要是經由順位調控子SIE（-87）和CRE-2(-70)去誘導mcl-1基因的轉錄。為了探討這些順位調控子在生理上扮演的角色，我們製造了SIE和CRE-2突變的小鼠(Ｍcl-1mSC/mSC)。研究發現相較於野生型小鼠，突變小鼠的次級淋巴組織中CD8+ T細胞約減少了一半。同時也發現mcl-1的RNA和蛋白質表現量在突變小鼠的T細胞是比較少的，但是在B細胞中並沒有減少的現象。這些結果顯示mcl-1基因轉錄的調控在T細胞和B細胞中可能是不同的。為了更近一步的研究可能的機制，我們使用電泳速度變動分析法(EMSA)來測試T細胞和B細胞核萃取蛋白質與含有mcl-1基因啟動子片段的DNA探針反應後，比較其蛋白質核酸結合的活性有無差異。我們發現在T細胞和B細胞中在mcl-1基因啟動子-97到-65片段，都可以偵測到SIE和CRE-2 complex的形成，同時SIE和CRE-2 complex中分別具有PU.1和phsphorylated CREB。另外，我們也發現在B細胞中，mcl-1啟動子上-156位置的順位調控子(AP2-like element)上會形成B細胞較多結合的蛋白質核酸複合體(B cell enriched binding complex, BEB complex)。綜合突變小鼠的表現型分析，以及相對於T細胞，在B細胞中具有較多的BEB complex結合於mcl-1啟動子上，因此我們推測在B細胞中mcl-1基因的轉錄可能主要是受到BEB complex的調控，而在T細胞中則主要是受到SIE和CRE-2順位調控子的調控。未來尚需要更多的實驗去驗證以上的結論。

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.