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 fi lament protein, is transcriptionally induced with a late time course w hen nerve growth factor stimulates PC12 cells to differentiate into ne urons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiate d and nonneuronal cells. Nerve growth factor-induced derepression of p eripherin gene expression is associated with alterations in proteins b inding to a GC-rich DNA sequence in the NRE as detected by the DNA ele ctrophoretic mobility shift assay (EMSA). We have utilized DNA affinit y chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identit y with NF1-L, a member of the CTF/NF-1 transcription factor family. Th is protein forms a single complex when incubated with the NRE probe us ing EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixin g the purified protein with the flow-through from the DNA affinity col umn, thereby demonstrating that protein-protein interactions are invol ved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analys is confirm that NF1-L, or a closely related family member, is the DNA- binding protein component of the multiprotein complex at the NRE.