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

为了揭示DMAHP/Six5在分子水平上参与肌强直性营养不良（DM）的发病机制，我们澄清了以下几点。(1)我们确定了两个骨骼肌、心脏和胚胎共有的转录起始位点和一个早期胚胎特异性的转录起始位点（El1）。所有这些位点都位于CTG重复序列的下游。(2)我们分析了P19细胞中DMAHP/Six5基因的转录调控元件，鉴定出3个Sp1/Sp3位点为正调控元件和2个负调控元件，其中一个与未知因子结合。(3)为了鉴定DMAHP/Six5蛋白的靶基因，我们构建了组成型活性VPl6-Six5和组成型抑制eng6 -Six5。本构的主动Six5如预期的那样发挥作用，但本构的压抑Six5却没有。利用微阵列技术将含有VP16-Six5的腺病毒载体感染到培养细胞中，筛选可能的靶基因。(4)肌原启动子已被确定为Six1、Six4蛋白的靶基因。我们发现启动子可以被Six5激活。Eya基因的共转染大大增强了其活化。这一结果提示了对糖尿病发病机制的新认识，即Six5的表达减少导致与Eya的协同作用减弱，从而导致肌生成素的表达减少。这可能导致骨骼肌发育不成熟。

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.