Heme is a cofactor for many important proteins that act in multiple cellular processes including aerobic respiration, biosynthesis, and small molecule processing. Previously, it has been shown that heme binds to regulatory and signaling proteins such as Hap1 in yeast, and Bach1 and Rev-erbα in mammals and controls their transcriptional activity. Recently, our studies investigated the regulatory role of heme in JmjC-domain-containing protein Gis1. Analysis of lacZ reporter driven by a PDS (post diauxic shift) element showed a significant increase in Gis1 transcriptional activity under high heme concentration. Spectrophotometric analysis demonstrated direct interaction of heme with Gis1 and JmjN/C domain of Gis1 mammalian homologous JMJD2B. Given that a high number of unique interaction partners have been identified for Gis1 previously, it is conceivable that Gis1-interacting proteins can mediate heme regulation of Gis1 activity. By performing pull-down assay followed by mass spectrometry, in vitro, we identified a group of proteins that interact with Gis1 under high heme concentration. Knocking out of encoding genes of the identified interacting proteins led to a substantial decrease in heme activation of Gis1. Our studies indicate that Gis1 transcriptional activity is heme dependent, and Gis1-interacting proteins can promote the heme activation of Gis1 activity.