There are fundamental gaps in understanding how regulatory proteins sense changes in oxygen and heme levels and promote cellular responses. Such gaps make it difficult to design effective strategies to treat diseases associated with hypoxia or heme dysregulation. The long- term goal is to better understand the molecular mechanisms by which the JmjC domain- containing proteins sense and promote cellular responses to changes in oxygen or heme levels. The objective of this application is to elucidate the molecular events underlying oxygen and heme regulation of Gis1 activity. The yeast Gis1 protein is a member of the JHDM3/JMJD2 subfamily of demethylases and a DNA-binding transcriptional regulator. Recent evidence shows that Gis1 is regulated by both oxygen and heme; Gis1 exemplifies a new class of regulators mediating heme and oxygen signaling. The central hypothesis is that the Gis1 JmjC domain cooperates with other domain(s) and proteins to sense and respond to changes in oxygen or heme levels and alter protein stability, protein-protein and protein-DNA interactions, thereby promoting oxygen and heme signaling. The rationale for the proposed work is that understanding how heme and oxygen regulate Gis1 can provide novel insights into how mammalian JmjC domain-containing proteins can be regulated by heme and oxygen and how their activity can be modulated to improve health. Guided by strong preliminary data, this central hypothesis will be tested by pursuing two specific aims: (1) Define the Gis1 modules mediating oxygen and heme signaling and characterize the direct interaction between Gis1 and heme; and (2) Characterize the effects of oxygen and heme on Gis1 protein interaction and DNA binding. Under the first aim, mutagenesis studies will be carried out to pinpoint Gis1 modules and residues critical for mediating oxygen and heme signaling in live cells, and in vitro studies will be carried out to examine the direct interaction between Gis1 and heme. Under the second aim, the effect of heme on Gis1 DNA binding will be examined, and purified Gis1 complexes will be characterized to determine the effect of oxygen and heme on Gis1 protein-protein interactions. The approach is innovative because it represents a new and substantive departure from the status quo in the studies of oxygen and heme signaling and of JmjC domain-containing demethylases. The proposed research is significant, because it is expected to advance the understanding of how the JmjC domain-containing proteins sense and respond to changes in oxygen and heme levels.