Dissecting the Role of Heme in Regulating Yeast and Mammalian KDM4 Histone Demethylases




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Heme (Iron protoporphyrin IX) is an essential biological molecule and an important metallonutrient for organisms ranging from bacteria to humans. It is also a signaling molecule that modulates the activities of diverse regulatory proteins. Recent studies have identified a novel class of heme-regulated proteins- the JmjC domain-containing proteins, constituted by the yeast Gis1, an ortholog of the human KDM4 proteins. Heme regulates the transcriptional and demethylase of activities of Gis1. The KDM4 subfamily of proteins containing the JmjC domain, function as a-ketoglutarate- and Fe (II)-dependent histone demethylases. Histone lysyl demethylases (KDMs) play important roles in chromatin remodeling and gene regulation by catalyzing demethylation of the lysine residues on the N-terminal tails of histones. The objective of this research was to dissect the molecular events underlying the heme regulation of Gis1 and KDM4 proteins. The JmjN/C domains of Gis1 and KDM4A/B/C share high homology, and the C-terminal domains of KDM4s possess the PHD zinc fingers. Here, using various biochemical methods, we showed that heme binds to purified KDM4 A/B/C proteins containing only the JmjN/C domain. Interestingly, we identified that heme stimulates the histone demethylase activities of KDM4 A and C proteins, containing only the JmjN/C domain and full length Gis1, but not KDM4B. Additionally, the C-terminal regions of KDM4s like Gis1 could confer heme regulation when fused to an unrelated transcriptional activator. Furthermore, a biochemical pulldown study coupled with mass spectrometry identified 147 heme-regulated protein interactors of Gis1. These proteins include a significant number of heterocyclic compound binding proteins, Ubl conjugated proteins, metabolic enzymes and acetylated proteins. These results demonstrate a previously uncharacterized heme-regulated protein complex formation of Gis1 and likely other KDM4s to sense metabolic and nutritional conditions and transduce signals to downstream regulatory pathways.



Heme, Enzymes, Protein-protein interactions, Yeast



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