Fujimoto Katsumi, Hamaguchi Hidenori, Hashiba Takafumi, Nakamura Tadahiro, Kawamoto Takeshi, Sato Fuyuki, Noshiro Mitsuhide, Bhawal Ujjal K, Suardita Ketut, Kato Yukio
Department of Dental and Medical Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan.
Int J Mol Med. 2007 Jun;19(6):925-32.
Dec2, a member of the basic helix-loop-helix (bHLH) superfamily, has been shown to function as a transcriptional repressor and is implicated in cell proliferation and differentiation. In addition, Dec2 transcripts exhibit a striking circadian oscillation in the suprachiasmatic nucleus. To identify the molecular mechanisms by which Dec2 regulates gene expression, we carried out structure-function analyses. Gel retardation and luciferase assays showed that Dec2, as well as its related protein Dec1, preferentially binds to class B E-box elements (CACGTG) as a homodimer and represses the transcription of target genes in a histone deacetylase (HDAC)-dependent manner. Functional studies with the GAL4-DNA binding domain fusion proteins identified the domain responsible for the repression activity of Dec2 in its C-terminal region, which is also necessary to recruit HDAC1. In addition, the basic and HLH domains of Dec2 were required for DNA binding and homodimerization, respectively. In contrast, Dec proteins repressed a MyoD-activated promoter activity of muscle creatine kinase gene through class A E-box in an HDAC1-independent manner. Dec2 formed a heterodimer with MyoD through the basic and HLH domains. Consistent with this, both the basic and HLH domains were required for the ability of Dec2 to inhibit the transcriptional activity of MyoD. These findings indicate that Dec2 employs multiple mechanisms, including DNA-binding and protein-protein interactions, to achieve E-box-dependent transcriptional repressions.
Dec2是碱性螺旋-环-螺旋（bHLH）超家族的成员之一，已被证明具有转录抑制因子的功能，并与细胞增殖和分化有关。此外，Dec2转录本在视交叉上核中呈现出显著的昼夜节律振荡。为了确定Dec2调节基因表达的分子机制，我们进行了结构-功能分析。凝胶阻滞和荧光素酶检测表明，Dec2及其相关蛋白Dec1作为同二聚体优先结合B类E盒元件（CACGTG），并以组蛋白去乙酰化酶（HDAC）依赖的方式抑制靶基因的转录。利用GAL4-DNA结合结构域融合蛋白进行的功能研究确定了Dec2 C端区域中负责其抑制活性的结构域，该结构域也是招募HDAC1所必需的。此外，Dec2的碱性结构域和HLH结构域分别是DNA结合和同二聚化所必需的。相比之下，Dec蛋白以HDAC1非依赖的方式通过A类E盒抑制肌肉肌酸激酶基因的MyoD激活启动子活性。Dec2通过碱性结构域和HLH结构域与MyoD形成异二聚体。与此一致的是，Dec2抑制MyoD转录活性的能力需要碱性结构域和HLH结构域。这些发现表明，Dec2采用多种机制，包括DNA结合和蛋白质-蛋白质相互作用，来实现E盒依赖的转录抑制。
