Two polypyrimidine tracts in the nitric oxide synthase 2 gene: similar regulatory sequences with different properties

We reported previously that the polymorphic polypyrimidine CCTTT-microsatellite in the regulatory region of nitric oxide synthase 2 (NOS2) bound nuclear proteins in vitro. In the present work, we aimed to characterize and investigate a potential regulatory role of the CCTTT-microsatellite in NOS2 expression. Therefore, we performed gel-shift, S1-nuclease, and chromatin immunoprecipitation (ChIP) assays. In vitro experiments showed that the microsatellite formed triplex-DNA both with and without superhelical constraint. We also found that the CCTTT-microsatellite and an apparently similar CT-repeat in the first intron of NOS2 were specifically cleaved by S1-nuclease, when cloned into a supercoiled plasmid. In vitro data suggested that the CCTTT-microsatellite bound both polypyrimidine tract-binding protein (PTBP1) and heterogeneous nuclear ribonucleoprotein K (hnRNPK). On the contrary, ChIP revealed binding of PTBP1 and hnRNPK rather to the CT-repeat in the first intron than to the CCTTT-microsatellite. Enrichment for RNA polymerase II and acetylated histones H3 and H4 was also detected at the intronic site. We suggest that both PTBP1 and hnRNPK binds the single strand of the triplex-DNA formed at the CT-repeat in the first intron and that this interaction could be involved in the regulation of NOS2 expression.