摘要
粒·巨噬细胞集落刺激因子(Granulocyte macrophage colony-stimulating factor,GM-CSF)是主要的造血生长因子之一,在造血调控和免疫调节中发挥重要作用。GM-CSF基因表达受转录水平和转录后水平调控,但主要受转录水平的调控。bcl10是从粘膜相关淋巴组织(mucosa-associated lymphoid tissue,MALT)淋巴瘤的染色体转位中发现的基因,这种染色体转位使整个bc110基因在染色体上受到Ig增强子的控制,进而导致其超量表达。BCL10在淋巴细胞抗原受体介导的NF-κB激活过程中是必需的,能调节细胞凋亡和NF-κB信号转导。本论文在构建含bcl10基因哺乳动物表达载体、构建GM-CSF启动子/增强子与luciferase嵌合载体、以及构建基于GM-CSF基础启动子的突变体(对GM-CSF基础启动子AP1、ETS1、NFAT、ETS1/NFAT转录因子作用位点的突变)的基础上,通过对白血病细胞株K526的转染,发现:
1)在K562细胞中,BCL10激活GM-CSF转录,Ionomycin促进BCL10对GM-CSF转录激活。
2)ETS1位点对于BCL10对GM-CSF的转录激活是必须的,细胞中正常水平的ETS1和AP1对GM-CSF的转录激活作用十分必要和重要,但细胞中过量的ETS1蛋白抑制BCL10对GM-CSF转录激活作用。
3)BCL10对GM-CSF的转录激活可能是通过一个独立于NF-κB途径进行的。
我们选取atxl基因的启动子作为研究对象。通过对启动子区域的5'和3'方向的缺失,构建了一系列表达载体,并转化Chlamydomonas reinharditii。通过对转化子的检测,确定了2处铁反应元件(FeREs)。即:AtxFeRE1在-529/-515(GTCGCACTGGCATGT)和AtxFeRE2在-300/-286(GCAGCGATGGCATTT)。二者有共同的序列:TGGCA.
Granulocyte macrophage colony-stimulating factor(GM-CSF)is a multipotent cytokine involved in the production and function of the hematopoietic cell.GM-CSF plays a major role in response to infection and physiological and pathological inflammatory processes.The expression of GM-CSF gene is controlled both in transcriptional and translational level,but primary regulated at transcriptional state.B-cell lymphoma 10(BCL10)gene was found in chromosomal dislocation in the cell of mucosa-associated lymphoid tissue(MALT)lymphoma. The chromosomal dislocation makes the bcl10 gene under the control of the enhancer of Ig, leading to over expression of bcl10.previous studies have shown that BCL10 involved in the activation of NF-κB signal pathway,and induce the cell apoptosis.In this study,on the base of producing the constructs including pcDNA1-BCL10,pGL2-GMCSFpr,pGL2-GMCSFpr/en, pGL2-GMCSFpr-basic,pGL2-GMCSFpr-mNFAT,pGL2-GMCSFpr-mETS1, pGL2-GMCSFpr-mAP1,and pGL2-GMCSFpr-mETS1/mNFAT,We transfer these plasmids to the cell of K562.The results demonstrate,
1)BCL10 activates the transcription of GM-CSF in K562,and improved by ionomycin.
2)The binding site of ETS1 is necessary for the transcriptional activation of GM-CSF by BCL10.The normal concentration of the ETS1 and AP1 are essential to the transcriptional activation of GM-CSF by BCL10,however,the over expression of ETS1 lead to repression of the transcriptional activation of GM-CSF by BCL10.
3)The transcriptional activation of GM-CSF by BCL10 is in a way independent from the NF-κB signal pathway.
We investigated the promoter region of atxl,which encodes a copper chaperone in response to iron deficiency induction.Deletion analysis of the promoter region from 5' and 3' ends revealed that the -532/-461 and -320 /-276 regions were necessary and sufficient for iron deficiency-inducible expression.Further deletion analysis showed that two of the Fe deficiency responsive elements(FeREs)localized within the -532/-511 and -306/-276 regions, in which AtxFeRE1 at -529/-515(GTCGCACTGGCATGT)and AtxFeRE2 at -300/-286 (GCAGCGATGGCATTT)had been identified respectively,with a conserved sequence of GNNGCNNTGGCATNT,differing from all known FeREs found in other organisms.
引文
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