PROTEIN-DNA INTERACTIONS DURING PHENOTYPIC DIFFERENTIATION

We have been studying the molecular mechanism of neuronal differentiat ion through which the multipotent precursor becomes limited to the fin al transmitter phenotype. Here we focused on the role of the 5' proxim al regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gen e in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient t o confer activity-dependent expression of the gene during neuro-differ entiation when it was tested using transient transfection assays of pr imary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, wit h nuclear proteins derived from phenotypically and ontogenetically dis tinct brain regions. Only a few low abundance protein-DNA complexes we re detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes d id not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify th e nature of the observed bands. Although both consensus NF1 and enkCRE 1(NF1) formed complexes with nuclear proteins derived from the striatu m and cortex at various ages, the appearance of the bands was not corr elated with ENK expression. Surprisingly, no complexes were detected i f other ENK-specific motifs were used as probes. We also tested nuclea r extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individ ual motifs. These experiments showed the formation of elaborate protei n-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-spe cific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which do es not express ENK, were used. Based on these observations, we conclud ed that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their bin ding proteins may be necessary to confer developmentally regulated, ce ll-specific expression of the ENK gene; and 2. Inducibility of the gen e by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.