DHR51 as a coordinator of heme biosynthesis and steroid hormone production to time metamorphosis in Drosophila melanogaster

Development from a juvenile to an adult animal is driven by pulses of steroid hormones released at precise developmental times. Inputs from the environment, timing cues, and nutritional factors are all coordinated to produce a steroid hormone pulse. Insects and in particular, the fruit fly Drosophila melanogaster, have long been used to study the actions of steroid hormones, how steroid hormones are made, and signaling pathways that regulate steroid hormone production. In Drosophila, the primary steroid hormone, ecdysone, is synthesized from cholesterol in a specialized endocrine gland called the prothoracic gland. Multiple highly expressed cytochrome P450 enzymes are involved in synthesizing ecdysone. Ecdysone is then released into the larval body to initiate developmental transitions such as larval molts and metamorphosis. Each cytochrome P450 enzyme requires heme as a cofactor to function. The prothoracic gland must also have a high demand for heme due to the high levels of cytochrome P450 enzymes. Mutations that disrupt late stages of heme biosynthesis cause the prothoracic gland to accumulate red autofluorescent heme precursors due to an attempt to increase heme biosynthesis. This unique accumulation of red autofluorescent heme precursors is only seen in two other tissues, which also happen to highly express cytochrome P450 enzymes, highlighting the importance of heme in the prothoracic gland for cytochrome P450 enzymes. Due to the connection between heme and cytochrome P450 enzymes, I hypothesize that heme is an important input into regulating ecdysone production in the prothoracic gland and that heme biosynthesis and ecdysone production are coordinately regulated during the final larval stage to produce a major ecdysone pulse to initiate metamorphosis. To understand the importance of heme and heme regulation in the prothoracic gland, my first aim was to identify a heme sensor that can detect when cellular heme levels are low and upregulate heme biosynthesis in response. My primary candidate for a heme sensor was the nuclear receptor DHR51, Drosophila hormone receptor 51, which is capable of reversibly binding heme in vitro. To determine whether DHR51 acts as a heme sensor, I used qPCR to determine that loss-of-DHR51 attenuated the expression of the rate-limiting enzyme in the heme biosynthesis pathway when cellular heme levels were low. RNA-Seq and heme measurements of DHR51-RNAi larvae provided evidence that loss-of-DHR51 disrupted heme homeostasis, lowering cellular heme levels. However, I was unable to determine whether heme binding is relevant in vivo and whether DHR51 functions as a heme sensor as predicted. In addition to DHR51’s apparent role in maintaining heme homeostasis, DHR51 is also necessary for ecdysone production. qPCR and RNA-Seq identified that DHR51-RNAi in the prothoracic gland reduced the expression of most of the ecdysone biosynthetic enzyme genes, which led to a reduced ecdysone titer that was measured with an ecdysone enzyme immunoassay. I provided evidence that DHR51 regulated ecdysone production through the circadian rhythm, the day-night cycle, as DHR51-RNAi disrupted the expression of core circadian rhythm genes. I commissioned the production of a DHR51 antibody that can be used in future chromatin immunoprecipitation experiments to identify direct target genes of DHR51 to determine whether DHR51 binds to heme biosynthetic genes, circadian rhythm genes, or ecdysone biosynthetic genes. Since heme and ecdysone are primarily produced during the night, I propose that DHR51 coordinates heme biosynthesis and ecdysone production in the prothoracic gland via the circadian rhythm.