NF1-L Is the DNA-binding Component of the Protein Complex at the Peripherin Negative Regulatory Element

doi:10.1074/jbc.270.12.6975

NF1-L Is the DNA-binding Component of the Protein Complex at the Peripherin Negative Regulatory Element (*)

From the Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

^¶ 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.

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

↵^§ Supported by the Medical Scientist Training Program at Vanderbilt University.
↵* 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.
↵¹ 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.
↵²L. Chang, and M. Thompson, unpublished results.
- Received October 28, 1994.
- Revision received December 27, 1994.