Abstract
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'-AATTACTCACATCC-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.
