Identification of a novel cis-acting positive element responsible for the cell-specific expression of the NK-1 homeobox gene

The Drosophila NK-1 homeobox gene belongs to the NK-1 class that includes a large number of vertebrate homeobox genes and is shown to be expressed in specific muscle founder cells and a subset of neuronal cells in the ventral nerve cord during embryogenesis. To determine the cis-acting regulatory elements controlling the cell-specific expression of NK-1, we measured transiently expressed chloramphencol acetyl transferase (CAT) reporter gene activities from transfected C₂C₁₂, myoblasts and NG108-15 neuroblastoma cells using various CAT constructs containing different 5' upstream regions of NK-1. From the initial analysis of 3.9 kb of the 5' upstream region, we have found that the regions from -1865 to -476 and from -476 to +100 contained strong negative and positive regulatory elements, respectively. Within the positive cis-acting region an 86-bp DNA fragment (from -435 to -350) was sufficient to activate the reporter gene in C₂C₁₂, cells, whereas additional regions (from -157 to -28 and from -510 to -425) were required for optimal activity in NG108-15 cells. Gel shift and DNaseI footprinting assays have defined a plausible binding site for C/EBP, 5'-TTTCGCAAG-3' (-424 to -416), and a novel binding site for unknown factors, 5'-AATTAC-TCACATCC-3' (-370 to -357). Further mutation analysis has revealed that the novel binding sequence for unknown factors is necessary and sufficient for transcriptional activity for reporter gene expression in C₂C₁₂, myoblast cells in an orientation-independent manner.