MnSOD Chromatin Structure

Our studies on the nuclear encoded, mitochondrially localized MnSOD gene have over the past ten years uncovered some exciting mechanisms underlying the regulation of this potent anti-oxidant protein by pro-inflammatory mediators. We have demonstrated that this CAT and TATA-less gene is highly regulated in a variety of tissues at the transcriptional level. By employing a strategy which attempts to evaluate gene regulation in as close to the in vivo situation as possible, we first analyzed the chromatin structure of the MnSOD by identifying the location of numerous DNase I hypersensitive sites (HS) flanking and within the gene. These experiments employed both low and novel high resolution analyses of chromatin structure which helped pinpoint the location of trans-acting factor binding sites.  The left upper panel below illustrates a classic analysis of DNase I hypersensitive (HS) sites as viewed at low resolution on a standard 1% agarose gel.  The right panel shows studies where the same gel system is employed but the a single site, HS site 1, is examined more closely by indirect end-labeling with a probe near the region of interest.  This demonstrates the more complex internal architecture of a hypersensitive site.

We then developed methodology to analyze hypersensitive sites at high resolution using a Metaphore and Nusieve agarose gel systems which afford the ability to reproducibly fractionate DNA fragments differing in size by only 100 bp.  These results shown below illustrate the existence of alternating regions of DNase I cleavage and protective regions indicative of transcription factor footprints. It also shows that gene activation by LPS causes changes in the chromatin structure with the appearance of HS sites 1-5 and 1-6.  The diagram on the left below illustrates our interpretation of the our data.