Han Liqun, Wong Dona Lee, Tsai Guochuan, Jiang Zhichun, Coyle Joseph T
Laboratory of Molecular and Psychiatric Neuroscience, Department of Psychiatry, Harvard Medical School and McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA.
Brain Res. 2007 Sep 19;1170:1-12. doi: 10.1016/j.brainres.2007.07.017. Epub 2007 Jul 17.
The expression of glutamate carboxypeptidase II (GCP II) is reduced in selective brain regions in schizophrenic patients. To investigate transcriptional mechanisms regulating the human GCP II gene, a 3460 bp DNA fragment comprised of the proximal 3228 bp of 5' untranscribed sequence and first 232 bp of 5' UTR portion of this gene was cloned into the mammalian luciferase reporter gene vector pGL3-Basic. Transfection assays in human astrocyte-derived SVG and human prostate tumor-derived LNCaP cells demonstrated that constructs with 3460, 1590 and 761 bp portions of 5' region of human GCP II gene were able to drive the luciferase reporter gene. Additional deletion constructs showed that in the SVG cell line, constructs with 511 and 411 bp of GCP II gene fragments yielded highest transcriptional activity, with declining activity upon further removal of 5' sequences. 15 bp of the promoter 5' to a 225 bp GCP II fragment were essential for luciferase expression. Thus, in the SVG cells, the proximal 240 bp of the human GCP II promoter (232 bp of the 5' UTR and 8 bp of 5' untranscribed sequences) may represent the core promoter. Further, while a LyF-1 site lies within and overlaps a transcription start site in the 15 bp sequence, site-directed mutagenesis shows that LyF-1 is not the transcription initiator for the "TATA and CAAT" box lacking GCP II gene in the SVG cells. Finally, pattern differences in GCP II gene promoter expression in SVG and LNCaP cells suggest that sequences beyond 240 bp may be important for tissue-specific GCP II expression.
谷氨酸羧肽酶II（GCP II）在精神分裂症患者的某些特定脑区表达降低。为了研究调控人类GCP II基因的转录机制，将一个3460 bp的DNA片段（由该基因5'非转录序列的近端3228 bp和5'UTR部分的前232 bp组成）克隆到哺乳动物荧光素酶报告基因载体pGL3-Basic中。在人星形胶质细胞来源的SVG细胞和人前列腺肿瘤来源的LNCaP细胞中进行的转染实验表明，包含人类GCP II基因5'区域3460、1590和761 bp部分的构建体能够驱动荧光素酶报告基因。进一步的缺失构建体表明，在SVG细胞系中，含有511和411 bp GCP II基因片段的构建体产生最高的转录活性，随着5'序列的进一步去除，活性下降。在一个225 bp GCP II片段上游5'的15 bp启动子序列对于荧光素酶表达至关重要。因此，在SVG细胞中，人类GCP II启动子的近端240 bp（5'UTR的232 bp和5'非转录序列的8 bp）可能代表核心启动子。此外，虽然一个LyF-1位点位于15 bp序列内且与转录起始位点重叠，但定点诱变表明LyF-1不是SVG细胞中缺乏“TATA和CAAT”框的GCP II基因的转录起始因子。最后，SVG和LNCaP细胞中GCP II基因启动子表达的模式差异表明，240 bp以外的序列可能对GCP II的组织特异性表达很重要。
