ABSTRACT

The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor stimulates PC12 cells to differentiate into neurons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiated and nonneuronal cells. Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a GC-rich DNA sequence in the NRE as detected by the DNA electrophoretic mobility shift assay (EMSA). We have utilized DNA affinity chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. This protein forms a single complex when incubated with the NRE probe using EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixing the purified protein with the flow-through from the DNA affinity column, thereby demonstrating that protein-protein interactions are involved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analysis confirm that NF1-L, or a closely related family member, is the DNA-binding protein component of the multiprotein complex at the NRE.

FOOTNOTES

*

This work was supported in part by Public Health Service Grant NS-30943 from the NINDS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by the Medical Scientist Training Program at Vanderbilt University.

¶

Recipient of a Research Career Development Award from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Cell Biology, C-2210 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-0337; Fax: 615-343-4539.

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The abbreviations used are: NGF, nerve growth factor; NRE, negative regulatory element; bp, base pair(s); EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; PPRS, perfect palindrome mutant repressor site oligonucleotide; WTRS, wild-type repressor site oligonucleotide; CV, column volumes.

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L. Chang, and M. Thompson, unpublished results.

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