Transcriptional regulation of the human ADP-ribosylation factor 5 (ARF5) gene

Hybridization of a blot containing 50 human RNAs with an ADP-ribosylation f actor 5-specific (ARF5) oligonucleotide probe revealed that the ARF5 gene i s expressed in all tissues: however, the level of expression varies signifi cantly with highest levels in pancreas, pituitary gland, and placenta. The 5'-flanking region of the human ARF5 gene lacks a TATA or CAAT box and is h ighly GC-rich. Primer extension analysis indicates that transcription initi ates at a discrete site 62 bp 5' to the start of translation: however, the sequence surrounding the transcription initiation sits does not resemble th e initiator elements described for other TATA-less genes. Transient transfe ction of. ARF5/luciferase deletion constructs into human IMR-32 neuroblasto ma cells revealed that sequences within 169 bp of the transcription initiat ion site were necessary for full expression. Two GC boxes within this regio n were modified by site-directed mutagenesis and found to be critical for e xpression of the reporter constructs. Electrophoretic mobility-shift assays demonstrated specific DNA/protein complexes could be formed with oligonucl eotides containing each of the GC boxes and these complexes could be effect ively competed by oligonucleotides containing either ARF5 Sp1 site or by an oligonucleotide containing a previously characterized Sp1-binding sequence . The level of ARF5 gene expression therefore, is dependent upon Sp1 or an Sp1-like factor but does not rely upon a canonical initiator element for ac curate transcription initiation. (C) 1999 Elsevier Science B.V. All rights reserved.