Chromatin Structure and In Vivo Footprinting Studies on Murine and Human iNOS

Nitric oxide (NO) has been implicated in a variety of normal and pathological situations, including vasodilation, neurotransmission, sepsis, as well as ischemia-reperfusion injury. There are currently three nitric oxide synthases (NOS) capable of generating NO, a neuronal form (nNOS) and an endothelial form (eNOS), which are constituitively expressed under most circumstances as well as an inducible form (iNOS).

iNOS has been strongly associated with the anti-bacterial and viral activity of monocytes in rodent cells, a phenomenon not yet documented in human cells. Although the exact physiological role of iNOS is not clearly been defined in for example pulmonary or hepatic cells, we have been attempting to understand the regulation of the human gene in these tissues.

Our studies on the murine and human iNOS genes in murine macrophages and astrocytes and human pulmonary and biliary epithelial cells have employed a strategy similar to that for MnSOD. In vivo footprinting studies of the murine iNOS promoter have identified the location of basal factor binding sites within the proximal promoter as well as inducible protein contacts. The studies in the murine system include a comparison of iNOS expression in macrophages with that in neuronal astrocyte cultures, which we have shown to have similar regulatory responses. Coupled with our gene regulation studies we have also begun an investigation of iNOS mRNA stability in murine astrocytes, based on the extensive evidence implicating this molecular mechanism in regulation of iNOS mRNA levels.

Our studies on the human iNOS gene were performed in collaboration with Dr. Sarah Chesrown at the University of Florida and Drs. Tim Billiar and David Gellar at the University of Pittsburgh. Transcription of the human iNOS gene is regulated by cellular stimulation with inflammatory cytokines in a tissue and species specific manner. To determine if differences in cytokine induced mRNA levels in human pulmonary epithelial cells (A549) or hepatic biliary epithelial cells (AKN-1) are the result of different protein/DNA regulatory mechanisms, we identified cytokine induced changes in chromatin structure by mapping DNase I hypersensitive (HS) sites in 13 kb of the 5’ flanking region. Our analysis revealed both constitutive and inducible HS sites in an overlapping yet cell-type specific pattern.

We then examined one region of chromatin that contained both constitutive and cytokine induced HS sites for potential DNA/protein interactions using dimethyl sulfate (DMS) in vivo footprinting and ligation-mediated PCR. We identified four potential protein binding sites at this location in the human iNOS promoter in both liver and lung cells. The in vivo footprinting autoradiogram below depicts sopme of this data and the figure on the left summarizes our results for this region of the humn iNOS promoter.

Three of these in vivo footprints are present in both control and cytokine-treated cells and map within a constitutive HS site. The remaining footprint appears only in response to cytokine treatment and maps to an inducible HS site. These studies illustrate a portion of the molecular mechanisms controlling the transcriptional regulation of the human iNOS gene at single nucleotide resolution in a cell/tissue specific manner. In vivo footprinting studies at -5 kb have revealed the interaction of numerous basal factors in both cell types. As part of our collaborative efforts we will pursue in vivo footprinting studies on the remainder of the chromatin hypersensitive sites and intiate the cloning of any novel factors.