Identification and characterization of a heme responsive element in the hrg-1 promoter

Despite its biological significance, little is known about how animals sense and respond to heme to maintain homeostasis. C. elegans is a heme auxotroph, which makes it an excellent model to identify and dissect heme homeostasis pathways. Using C. elegans we have identified HRG-1, a vesicular heme transporter that is transcriptionally upregulated when environmental heme is low. The current study seeks to address how hrg-1 is regulated by heme. Here, we show that a putative 23 base pair (bp) heme-responsive element (HRE) and GATA-binding motifs are necessary for heme-dependent regulation of hrg-1. The HRE comprises both enhancer and repressor elements and works in conjunction with ELT-2 to regulate hrg-1 expression. We propose that the HRE could be used as a molecular tool in C. elegans to tightly regulate internal gene expression by modulating environmental heme. Our ultimate goal is to identify the trans-acting factor to eventually create a whole animal sensor for monitoring organismal heme homeostasis

FUNCTIONAL INSIGHTS INTO HRG-1-MEDIATED HEME TRANSPORT

Heme is an essential cofactor involved in various biological processes including oxygen transport, xenobiotic detoxification, oxidative metabolism, gas sensing, circadian rhythm, signal transduction, microRNA processing and thyroid hormone synthesis. Heme is also an essential nutrient for parasites and is the major dietary iron source for humans. Despite our extensive understanding of the mechanisms of heme synthesis and degradation in eukaryotes, little is known as to how heme is transported and trafficked in eukaryotes. Recently, CeHRG-1 and CeHRG-4 were identified as the first bona fide heme importers/transporters using the heme auxotroph, Caenorhabditis elegans. To gain mechanistic insights into the heme transport function of HRG-1-related proteins, we conducted a structure-function analysis of CeHRG-1 and CeHRG-4 by exploiting yeast mutants that are genetically defective in heme synthesis. Our studies reveal that HRG-1-related proteins transport heme across membranes through the coordinated actions of strategically placed amino acids that are topologically conserved in both, the worm and human proteins. To further dissect the functional elements that dictate their intracellular localization, we generated a series of chimeras by swapping the amino and carboxy terminal segments of CeHRG-1 and CeHRG-4. Our analysis in yeast and mammalian cells demonstrate that the C-terminal domains are essential for membrane localization of the protein, while the N-terminal domains are important for proper function, and plausibly multimerization of HRG-1-related proteins. Currently, there are no pharmacological means to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We, for the first time, developed and executed a high-throughput screen of 233,360 compounds, to identify potential antagonists of HRG-1-related proteins by utilizing parasite heme transporters as the primary screening bait. Subsequent study in parasites will provide novel drug candidates against helminths that infect humans, livestock, and plants, as well as against genetic disorders of heme and iron metabolism in humans. Taken together, results from our studies will significantly advance novel functional and therapeutic insights into HRG-1 mediated heme transport in health and disease

LONG-RANGE SIGNALING AT THE INTESTINAL-NEURAL AXIS PROMOTES ORGANISMAL HEME HOMEOSTASIS IN C. ELEGANS

Metazoans synthesize and regulate intracellular heme in a cell autonomous manner although genetic evidence in vertebrates suggests that cell non-autonomous mechanisms may exist at the organismal level. In C. elegans, a heme auxotroph, extraintestinal tissues are intrinsically dependent on the intestine, which acquires dietary heme for sustenance, supporting the concept that intestinal heme status must be coordinated at the systemic level to regulate whole-organism heme homeostasis. Here we show, by conducting a functional genome-wide RNAi screen in an intestinal-restricted heme sensor worm, that an interorgan heme signaling pathway exists and that >30% of the genes identified from the RNAi screen altered heme homeostasis in the intestine even though these genes are not expressed in the intestine. The biological basis for this signaling is underscored by HRG-7, a cathepsin protease-like protein secreted by the intestine and internalized by distally-located neurons. HRG-7 is specifically secreted from the intestine during heme limitation and hrg-7 depletion causes embryonic lethality concomitant with a heme deficiency response. Reciprocally, neuron-to-intestine heme signaling is mediated by the bone morphogenic protein homolog DBL-1, which recapitulates hrg-7 deficiency when depleted. Remarkably, depletion of both genes simultaneously results in markedly enhanced growth and heme deficiency phenotypes, suggesting that bidirectional signaling between the intestine and neurons mediates systemic heme homeostasis. Our results have uncovered an unexpected role for a protease family member in long-range communication between organs at the intestinal-neural axis to regulate systemic heme homeostasis in metazoa. As humans have over thirty cathepsin and cathepsin-like proteases, several of which are secreted, we anticipate that these proteins may play analogous roles in mammalian biology