Neonatal jaundice is a condition that affects children throughout the world. Pathologic jaundice becomes a serious threat to the well being of neonates in the context of hemolytic disease, which is associated with increased bilirubin production. Our laboratories' approaches to this problem are to study bilirubin production, assess the efficacy of potential therapeutic agents that inhibit the activity of key enzymes, heme oxygenase isozymes 1 (HO-1) and 2 (HO-2) in the catalysis of heme to bilirubin, and use imaging to reveal in vivo transcription patterns. Expression of HO-1 in response to a variety of inducers, such as heme and heavy metals varies both in culture and in vivo. Metalloporphyrins (Mps), heme analogs, are clinically relevant inhibitors of HO enzyme activity, but some Mps have also been shown to increase HO-1 transcription and, in turn, may offset their therapeutic use. We have observed that the HO-1 transcriptional response to repeated treatments with the inducers are dampened in comparison to the initial response. These observations have led to the hypothesis that transcriptional activation of the HO-1 gene may lead to epigenetic changes that affect the ability of this gene to be transcribed in response to subsequent induction stimuli. This basic hypothesis and the potential mechanisms regulating these effects will be addressed in this study through three specific aims. First, we will use reporter cell lines (HO-1-luc) and a transgenic reporter mouse model, to characterize the short- and long-term effects of cadmium and heme on HO-1 transcription. These are known inducers of HO-1 transcription, which will be used as benchmarks for assessing the effects of Mps on HO-1 transcription. Second, we will subsequently identify region(s) of the HO-1 promoter, which mediate any of the observed long-term effects of Mp treatment. Third, we will then characterize these identified regions with respect to the presence of potential binding sites for nuclear factors. We have observed that after HO-1 gene activation there are increases in DNA methylation of the HO-1 promoter at a CpG rich region, which sits over the translational start site. This region will be targeted for studying differential binding of nuclear factors in active and repressed DNA. There are most likely significant long-term effects of activating any number of genes via approved and experimental chemotherapeutic strategies, and we will use the HO-1 -luc model to reveal mechanisms of transcriptional modulation subsequent to activation that may be involved in these long-term effects.
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