Wang Jianrong, Yen Andrew
Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853, USA.
Mol Cell Biol. 2004 Mar;24(6):2423-43. doi: 10.1128/MCB.24.6.2423-2443.2004.
The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.
维甲酸（RA）的作用机制与细胞和发育生物学、营养以及癌症化疗密切相关。已知RA可诱导伯基特淋巴瘤受体1（BLR1）基因的表达，该基因促使RA诱导的细胞周期停滞以及HL-60人髓母细胞白血病细胞的分化，从而推动了目前对RA对blr1表达的转录调控的分析。此处使用的经RA处理的HL-60细胞表达了除RXRγ之外的所有RA受体（RAR）和类视黄醇X受体（RXR）亚型（通过Northern分析检测）。用RAR和RXR选择性配体处理表明，RARα与RXRα协同作用以转录激活blr1表达。发现一个能够支持RA诱导HL-60细胞中blr1激活的5'侧翼区域在远端部分包含一个205 bp的序列，该序列对于RA的转录激活是必需的。在该序列内，DNase I足迹分析表明RA诱导一种核蛋白复合物与一个包含两个GT框的元件结合。电泳迁移率变动分析（EMSA）和超迁移分析表明该元件结合重组RARα和RXRα。没有RA时，既没有复合物结合也没有转录激活。两个GT框对于结合复合物都是必需的，并且任一GT框的突变都会导致RA的转录激活丧失。这种顺式作用的RAR-RXR结合元件响应RA激活转录的能力还取决于下游序列，在该元件与转录起始点之间的一个八聚体转录因子1（Oct1）位点和一个活化T细胞核因子（NFATc）位点，以及blr1基因转录起始点与第一个外显子之间的一个环磷酸腺苷反应元件结合因子（CREB）位点是必需的。这些位点中的每一个都结合其相应的转录因子。对经RA处理的细胞的核裂解物进行的转录因子-转录因子结合阵列分析表明有几个突出的RARα结合伙伴；其中，通过竞争EMSA、超迁移和染色质免疫沉淀分析确定Oct1、NFATc3和CREB2是该复合物的组成成分。RA上调了这三种因子的表达。总之，本研究结果表明RA诱导的blr1表达取决于一个新的RA反应元件。这个位于转录起始点上游约1 kb处的顺式作用元件由两个GT框组成，它们在一个核蛋白复合物中结合RAR和RXR，该复合物还包含与它们同源的下游共有结合位点结合的Oct1、NFATc3和CREB2。
