C EBP-RELATED SITES IN ADDITION TO A STAT SITE ARE NECESSARY FOR CILIARY NEUROTROPHIC FACTOR-LEUKEMIA FACTOR-DEPENDENT TRANSCRIPTIONAL ACTIVATION BY THE VASOACTIVE-INTESTINAL-PEPTIDE CYTOKINE RESPONSE ELEMENT/

The neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leuk emia inhibitory factor (LIF) regulate VIP gene expression through a cy tokine response element (CyRE) which interacts with members of the STA T transcription factor family. The CyRE STAT site is, however, insuffi cient to mediate full transcriptional activation by CNTF/LIF, suggesti ng that other sequences and nuclear proteins are also important. As C/ EBP proteins participate in the transcriptional effects of the related cytokine, interleukin-6, we investigated the role of possible C/EBP-b inding sites in the response of the VIP CyRE to CNTF/LIF. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, an d mutational analysis, three sites within the VIP CyRE were identified as C/EBP-related binding sites and shown to be important to CNTF/LIF- mediated transcriptional activation. The CyRE C/EBP-related sites inte ract with nuclear proteins from the human neuroblastoma cell Line, NBF L, including a novel, protein synthesis-dependent, nuclear protein com plex, induced by CNTF treatment, These nuclear proteins are not, howev er, recognized by antisera to known C/EBP proteins, Therefore, other n uclear proteins regulated by independent pathways act in concert with the JAK-STAT pathway to mediate CNTF/LIF regulation of VIP gene expres sion through the CyRE.