A NF-kappa B/Sp1 region is essential for chromatin remodeling and correct transcription of a human granulocyte-macrophage colony-stimulating factor transgene

The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb tran sgene containing the entire human GM-CSF gene has also been shown to displa y inducible, position-independent, copy number-dependent transcription in m ouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter an d activity assessed following the generation of transgenic mice. Of four mu tations introduced into the transgene promoter, only one, in an NF-kappaB/S p1 region, led to decreased induction of the transgene in splenocytes or bo ne marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, b ut not when transiently transfected into the same cell lines. The mutation alters the NF-kappaB family members that bind to the NF-kappaB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersens itive site that is normally generated at the promoter following T cell acti vation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-kappaB/Sp1 region plays a critical role i n chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.