Promoter analysis of human glutamate carboxypeptidase II.
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.
Han
Liqun
L
Laboratory of Molecular and Psychiatric Neuroscience, Department of Psychiatry, Harvard Medical School and McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA.
Wong
Dona Lee
DL
Tsai
Guochuan
G
Jiang
Zhichun
Z
Coyle
Joseph T
JT
D020121:5' Untranslated Regions; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D043425:Glutamate Carboxypeptidase II / Q000096:biosynthesis / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051740:Ikaros Transcription Factor / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000302:isolation & purification; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D020121:5' Untranslated Regions; D000954:Antigens, Surface; C497239:IKZF1 protein, human; D051740:Ikaros Transcription Factor; D012313:RNA; D008156:Luciferases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II
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