NF1-L IS THE DNA-BINDING COMPONENT OF THE PROTEIN COMPLEX AT THE PERIPHERIN NEGATIVE REGULATORY ELEMENT

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.