Transcriptional activation of the p34cdc2 gene by cdc2 promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.
Transcriptional activation of the p34cdc2 gene by cdc2 promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.
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胎鼠心室肌细胞中 cdc2 启动子结合因子/核因子-Y 对 p34cdc2 基因的转录激活。
DOI：
10.1161/01.res.82.2.251
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发表时间：
1998
影响因子：
20.1
通讯作者：
Zhu,H
中科院分区：
文献类型：
--
作者：
Liu,Q;Yan,H;Dawes,NJ;Lu,Y;Zhu,H
为了确定心肌终末分化如何受细胞周期控制基因的调节，我们研究了大鼠心肌中cdc 2的表达，发现与胎儿心室相比，新生儿心室中cdc 2 mRNA和蛋白水平降低，并且在青少年和成人心室中检测不到。为了进一步确定cdc 2下调是否归因于转录减少，使用逐渐截短的6.2-、1.8-、1.1-、0.7-和0.1-kb人cdc 2 5′侧翼区进行瞬时表达测定。所有五个片段激活报告基因在胎儿心肌细胞中的表达，并显着降低新生儿心肌细胞的活性。0.1 kb片段的活性为6.2 kb片段的65%。一个蛋白结合位点，其中包含一个倒置的CCAAT盒内的0.1 kb的片段，DNA酶I足迹测定，并命名为cdc 2启动子结合因子（CPBF）网站。点突变内的CPBF网站，废除CPBF结合显着降低0.1和6.2 kb的启动子活性。竞争实验和抗体超移实验表明，CPBF与转录因子核因子Y（NF-Y）相同或相关。在胎儿心肌细胞中，显性负性NF-Y突变体抑制了0.1-kb启动子活性。综上所述，我们的研究结果表明，心脏cdc 2表达下调出生后关闭时，达到少年阶段。cdc 2的一个0.1 kb的启动子片段包含了cdc 2在胎儿和新生儿心肌细胞中转录激活和抑制的主要信息。NF-Y或其相关因子在激活0.1kb cdc 2启动子中起关键作用。
—To determine how myocardial terminal differentiation is regulated by cell cycle control genes, we studied cdc2 expression in rat cardiac muscle and found that cdc2 mRNA and protein levels were reduced in neonatal compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression assay was performed using the progressively truncated 6.2-, 1.8-, 1.1-, 0.7-, and 0.1-kb human cdc2 5′ flanking regions. All five fragments activated reporter expression in fetal myocytes and were significantly less active in neonatal myocytes. The 0.1-kb fragment showed 65% of the activity of the 6.2-kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1-kb fragment by DNase I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1- and 6.2-kb promoter activities. Competition and antibody supershift assays suggested that CPBF was identical or related to the transcription factor, nuclear factor Y (NF-Y). The 0.1-kb promoter activity was suppressed by a dominant-negative NF-Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off when the juvenile stage is attained. A 0.1-kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation and suppression in fetal and neonatal myocytes, respectively. NF-Y or its related factor plays a critical role in activating the 0.1-kb cdc2 promoter.