Abstract
The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at -173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (-2660 to -2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E. (1992) Mol. Cell. Biol. 12,2501-2513). Here we define a distal positive element (DPE, -2445 to -2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements.
Highlights
Type [5, 6, 8, 11, 12]
Fine Deletion Mapping of the Distal Positive Region of the Peripherin Gene—Previously, a region between Ϫ2660 and Ϫ2308 of the peripherin 5Ј-flanking DNA was shown to be necessary for significant activity of the peripherin gene in response to nerve growth factor (NGF) [1]
A Distal Negative Element Is Necessary for Tissue-specific Expression of the Peripherin Gene—Since our results demonstrate that there is significant activity of the Ϫ2445-CAT peripherin reporter construct in 3T3 cells, this suggests that the negative regulatory element (NRE) element that represses peripherin activity in undifferentiated PC12 cells [1] is not sufficient to overcome the positive activity of the distal positive element (DPE) in non-neuronal cells
Summary
Cell Culture and Treatment—Stock cultures of PC12 cells were maintained as described previously [27]. Gel Retardation Assays (EMSAs)—The conditions for the DNA-protein binding reactions were essentially as described previously [1]. Reaction products were cut with HindIII, gel-purified, and subcloned into the HindIII site at the 5Ј boundary of the Ϫ2308-CAT construct. This introduces 10 bp of polylinker sequence between the PCR fragment and the peripherin sequence in the Ϫ2308-CAT plasmid. Clones containing a single mutation (I or II) or both mutations (I and II) were confirmed by dideoxynucleotide DNA sequencing, and the Ϫ2445 to Ϫ2308 HindIII insert was subcloned back into Ϫ2308-CAT to generate the mutant Ϫ2445-CAT constructs. Synthetic DNA Oligonucleotides—Oligonucleotides used in DNA mobility shift assays were flanked by HindIII restriction sites, synthesized on a Milligen Biosearch Cyclone Plus DNA synthesizer, and gelpurified
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