Department of Physiology and Biophysics, University of California, Irvine, D-346, Medical Sciences Building I, Irvine, CA 92697, USA.
Exp Physiol. 2009 Dec;94(12):1163-73. doi: 10.1113/expphysiol.2009.049692. Epub 2009 Aug 14.
The rat heart expresses two myosin heavy chain (MHC) isoforms, beta and alpha; these genes are arranged in tandem on the same chromosome. We have reported that an antisense (AS) beta RNA starts in the intergenic (IG) region between beta and alpha genes and extends to overlap the beta gene. We propose that in adult rats, both the alpha sense and IG betaAS RNA expression are activated by an IG bidirectional promoter and that the transcription of betaAS RNA interferes with the sense beta, resulting in low levels of beta mRNA and high levels of alpha, a phenotype seen in a typical rat heart. A previous report examined the activity of the betaAS promoter and showed that a 559 bp fragment of the betaAS promoter (-2285 to -1726; relative to alphaMHC gene start site) injected into rat ventricle was activated in control heart, and decreased significantly in response to hypothyroidism (propylthiouracil induced) and diabetes (streptozotocin induced) and increased in hyperthyroid rats (T(3) induced), similar in pattern to the endogenous betaAS RNA. In the present paper, we demonstrate with electrophoretic mobility shift analyses that ventricular nuclear proteins are interacting with a nuclear factor 1/CAAT-binding transcription factor 1 (NF1/CTF1) binding site, and a supershift assay indicates that the protein binding at this site is antigenetically related to the CTF1/NF1 factor. Moreover, a mutation of the CTF1/NF1 site within the 559 bp promoter region nearly abolished promoter activity in vivo in control, STZ- and PTU-treated rats. Based on these findings, we conclude that the NF1 site is critical to betaAS promoter regulation.
大鼠心脏表达两种肌球蛋白重链（MHC）同工型，β和α；这些基因在同一条染色体上串联排列。我们已经报道，反义（AS）β RNA 从β和α基因之间的基因间（IG）区域开始，并延伸到β基因重叠。我们提出，在成年大鼠中，α sense 和 IG betaAS RNA 的表达均由 IG 双向启动子激活，并且βAS RNA 的转录干扰了β sense，导致β mRNA 水平低而α水平高，这是典型大鼠心脏中所见的表型。先前的报告检查了βAS 启动子的活性，并显示出βAS 启动子的 559 bp 片段（-2285 至-1726；相对于αMHC 基因起始位点）注入大鼠心室在对照心脏中被激活，并且在甲状腺功能减退症（丙基硫氧嘧啶诱导）和糖尿病（链脲佐菌素诱导）时显著降低，并且在甲状腺功能亢进大鼠（T3 诱导）中增加，与内源性βAS RNA 的模式相似。在本文中，我们通过电泳迁移率移位分析证明，心室核蛋白与核因子 1/CAAT 结合转录因子 1（NF1/CTF1）结合位点相互作用，并且超迁移测定表明该位点的蛋白结合与 CTF1/NF1 因子具有抗原关系。此外，在 559 bp 启动子区域内 CT1/NF1 位点的突变几乎在对照、STZ 和 PTU 处理的大鼠体内完全消除了启动子活性。基于这些发现，我们得出结论，NF1 位点对于βAS 启动子调节至关重要。
