Pastorcic Martine, Das Hriday K
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
Brain Res. 2007 Jan 12;1128(1):21-32. doi: 10.1016/j.brainres.2006.10.056. Epub 2006 Nov 28.
DNA sequences required for the expression of the human presenilin 1 (PS1) gene have been identified between -118 and +178 flanking the major initiation site (+1) mapped in SK-N-SH cells. Several Ets sites are located both upstream as well as downstream from the +1 site, including an Ets motif present at -10 that controls 90% of transcription in SK-N-SH cells. However, in SH-SY5Y cells, transcription initiates further downstream and requires an alternative set of promoter elements including a +90 Ets motif. Ets2, ER81, ERM and Elk1 were identified by yeast one-hybrid selection in a human brain cDNA library using the -10 Ets motif as a bait. We have shown that ERM recognizes specifically Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments whether or not they contain the -10 Ets site. We have now searched for ERM interacting proteins by yeast two-hybrid selection in a human brain cDNA library using the C-terminal 415 amino acid of ERM as a bait. One of the interacting proteins was ZNF237, a member of the MYM gene family. It is widely expressed in different tissues in eukaryotes under several forms derived by alternative splicing, including a large 382 amino acid form containing a single MYM domain, and 2 shorter forms of 208 and 213 amino acids respectively that do not. We show that both the 382 as well as the 208 amino acid forms are expressed in SK-N-SH cells but not in SH-SY5Y cells. Both forms interact with ERM and repress the transcription of PS1 in SH-SY5Y cells. The effect of both C-terminal and N-terminal deletions indicates that the N-terminal 120 amino acid region is required for interaction with ERM in yeast, and furthermore single amino acid mutations show that residues 112 and 114 play an important role. The repression of transcription in SH-SY5Y cells also appears to require the N-terminal potion of ZNF237 and was affected by mutation of the amino acid 112. Data from electrophoretic mobility shift assays indicate that ERM and possibly ZNF237 interact with a fragment of the PS1 promoter.
已在SK - N - SH细胞中定位的主要起始位点（+1）侧翼-118至+178之间鉴定出人类早老素1（PS1）基因表达所需的DNA序列。几个Ets位点位于+1位点的上游和下游，包括位于-10处的一个Ets基序，它控制SK - N - SH细胞中90%的转录。然而，在SH - SY5Y细胞中，转录起始于更下游，并且需要一组不同的启动子元件，包括一个位于+90的Ets基序。通过酵母单杂交筛选，以-10 Ets基序为诱饵，在人脑cDNA文库中鉴定出Ets2、ER81、ERM和Elk1。我们已经表明，ERM特异性识别位于-10以及下游+90、+129和+165处的PS1启动子上的Ets基序，并且无论启动子片段是否包含-10 Ets位点，都能激活PS1转录。我们现在通过酵母双杂交筛选，以ERM的C末端415个氨基酸为诱饵，在人脑cDNA文库中寻找与ERM相互作用的蛋白质。其中一种相互作用蛋白是ZNF237，它是MYM基因家族的成员。它以几种通过可变剪接产生的形式在真核生物的不同组织中广泛表达，包括一种含有单个MYM结构域的382个氨基酸的大形式，以及分别为208和213个氨基酸的2种较短形式，后两种不含有MYM结构域。我们表明，382个氨基酸形式以及208个氨基酸形式在SK - N - SH细胞中表达，但在SH - SY5Y细胞中不表达。这两种形式都与ERM相互作用，并在SH - SY5Y细胞中抑制PS1的转录。C末端和N末端缺失的影响表明，N末端120个氨基酸区域是酵母中与ERM相互作用所必需的，此外，单个氨基酸突变表明第112和114位残基起重要作用。SH - SY5Y细胞中转录的抑制似乎也需要ZNF237的N末端部分，并且受第112位氨基酸突变的影响。电泳迁移率变动分析的数据表明，ERM以及可能的ZNF237与PS1启动子的一个片段相互作用。
