Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5.
Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5.
复制标题
DOI：
--
复制
发表时间：
1999-02
期刊：
影响因子：
4.6
通讯作者：
J. Reecy;Xuyang Li;M. Yamada;F. DeMayo;C. Newman;R. Harvey;R. Schwartz
中科院分区：
文献类型：
--
作者：
J. Reecy;Xuyang Li;M. Yamada;F. DeMayo;C. Newman;R. Harvey;R. Schwartz
Nkx2-5标志着最早可识别的心脏祖细胞，并在涉及谱系规范的诱导信号响应中被激活。Nkx2-5也在发育中的前肠、甲状腺、脾脏、胃和舌头中表达。一种阐明参与心脏发生的信号的方法是检查早期谱系标记（如Nkx2-5）的转录调控。我们产生了F0转基因小鼠，这些小鼠携带与lacZ报告基因相关的Nkx2-5侧翼序列。我们在Nkx2-5基因近10.7 kb处发现了多个调控区域。除了近端启动子外，我们还发现了第二个启动子和一个新的上游外显子，它们可以参与Nkx2-5转录的调控。虽然在正常发育中很少使用，但这种新的外显子可以通过几种方式拼接到Nkx2-5编码区，从而可能产生新的Nkx2-5蛋白亚型，其转录活性与野生型Nkx2-5相比大大降低。在编码外显子1和上游新外显子1a之间的3.5 kb外显子1内，发现了一个指导咽、脾、甲状腺和胃表达的增强子。外显子1a上游的两个或多个增强子能够在心脏新月中驱动表达，贯穿早期心管的整个心肌，然后是环心管的流出道和右心室。在exon1a的上游也有一个负性元件，它以复杂的方式与增强子相互作用，指导正确的空间表达。此外，Nkx2-5和GATA-4可以协同刺激潜在的自调节元件。我们的研究结果表明，一套复杂的相互作用的调控结构域调节Nkx2-5的转录。解剖这些元素可以揭示心脏诱导的基本特征以及心野内的正、负信号。
Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is activated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomach and tongue. One approach to elucidate the signals involved in cardiogenesis was to examine the transcriptional regulation of early lineage markers such as Nkx2-5. We generated F0 transgenic mice, which carry Nkx2-5 flanking sequences linked to a lacZ reporter gene. We identified multiple regulatory regions located within the proximal 10.7 kb of the Nkx2-5 gene. In addition to a proximal promoter, we identified a second promoter and a novel upstream exon that could participate in the regulation of Nkx2-5 transcription. Although used rarely in normal development, this novel exon could be spliced into the Nkx2-5 coding region in several ways, thereby potentially creating novel Nkx2-5 protein isoforms, whose transcriptional activity is greatly diminished as compared to wild-type Nkx2-5. An enhancer that directs expression in pharynx, spleen, thyroid and stomach was identified within 3.5 kb of exon 1 between the coding exon 1 and the novel upstream exon 1a. Two or more enhancers upstream of exon 1a were capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, then in the outflow tract and right ventricle of the looped heart tube. A negative element was also located upstream of exon1a, which interacted in complex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4. Our results demonstrate that a complex suite of interacting regulatory domains regulate Nkx2-5 transcription. Dissection of these elements should reveal essential features of cardiac induction and positive and negative signaling within the cardiac field.