Function of DMAHP/Six5 gene and the involvement in myotonic dystrophy

为了在分子水平上揭示DMAHP/Six 5在强直性肌营养不良（DM）发病机制中的参与，我们阐明了以下几点。(1)We鉴定了骨骼肌、心脏和胚胎共有的两个转录起始位点和早期胚胎特异的一个位点（E11）。所有这些位点都位于CTG重复序列的下游。这一观察排除了转录mRNA中的CUG重复序列导致异常剪接或翻译导致DM的可能性。(2)We分析了P19细胞中DMAHP/Six 5基因的转录调控元件，并鉴定了三个Sp1/Sp3位点为正调控元件和两个负调控元件，其中一个与未鉴定的因子结合。(3)To鉴定DMAHP/Six 5蛋白靶基因，构建了组成型活性VP 16-Six 5和组成型抑制性Eng-Six 5。组成型活性Six 5功能如预期，但组成型抑制Six 5没有。通过将含有VP 16-Six 5的腺病毒载体感染到培养的细胞中，使用微阵列进行可能的靶基因的筛选。（4）肌细胞生成素启动子是Six 1、Six 4蛋白的靶基因。我们发现启动子可以被Six 5激活。Eya基因的共转染大大增强了激活。这一结果提示了对DM发病机制的新见解，即Six 5表达减少导致与Eya的弱协同作用，从而导致肌生成素表达减少。这可能导致骨骼肌的不成熟。

To reveal the involvement of DMAHP/Six5 in pathogenesis of myotonic dystrophy (DM) at the molecular level, we clarified several points described below.(1)We identified two transcription start sites common to skeletal muscle, heart, and embryo and one site specific to early embryo (El1). All of these sites reside downstream of the CTG repeat. This observation excludes the possibility that the CUG repeat in the transcribed mRNA causes aberrant splicing or translation leading to DM.(2)We analyzed the transcription regulatory elements of the DMAHP/Six5 gene in P19 cells and identified three Sp1/Sp3 sites as positive regulatory elements and two negative regulatory elements, to one of which an unidentified factor binds.(3)To identify target genes of DMAHP/Six5 protein, we constructed constitutive active VPl6-Six5 and constitutive repressive Eng-Six5. The constitutive active Six5 functions as expected but the constitutive repressive Six5 did not. The screening of the possible target genes are ongoing using microarray by infecting adenovirus vector containing the VP16-Six5 into cultured cells.(4)The myogenin promoter has been identified as a target gene for Six1, Six4 protein. We found out that the promoter could be activated by Six5. Co-transfection of Eya genes greatly enhanced the activation. This result suggests a new insight into the DM pathogenesis that diminished expression of Six5 lead to weak cooperative action with Eya leading to reduced expression of myogenin. This might cause the immaturation of skeletal muscle.