Characterizing novel genes involved in steroidogenesis during the larval development in Drosophila melanogaster
Creators: Wang, Song
Publisher: name: University of Alberta Library
Year: 2022

<p>The primary steroid hormone in Drosophila melanogaster is called ecdysone, which is synthesized in the larval prothoracic gland (PG) during developmental processes. Ecdysone biosynthesis is significantly up-regulated at the beginning of the third larval instar, resulting in a major ecdysone pulse that triggers metamorphosis. Cytochrome P450 enzymes (P450) require heme as a cofactor and play a key role in catalyzing the conversion from dietary cholesterol to ecdysone. Since heme needs iron to function, there has been a link between cellular heme/iron homeostasis and ecdysone biosynthesis in Drosophila. <br>
My thesis focuses on two genes identified from two independent RNA interference (RNAi) screens carried out by previous lab members. The RNAi screens uncovered genes that may play essential roles in Drosophila development and heme biosynthesis. I will outline in two individual chapters how two genes of interest (RanBP3 and Su(var)2-10) impinge on ecdysone biosynthesis via different mechanisms.<br>
RanBP3-depletion animals showed a late-larval lethality, and interestingly, a porphyria-like phenotype in the prothoracic gland. Porphyrias are a group of eight rare metabolic diseases induced by disrupted heme biosynthetic pathway. I showed that the RanBP3 loss-of-function phenotype resulted from a disruption of heme biosynthesis and the accumulation of heme precursors. RanBP3 was further shown to play a vital role in the nuclear export of Iron-regulatory protein 1A (IRP1A), a central cellular iron sensor and regulator of iron homeostasis. I also examined how IRP1A is transported into the nucleus, which relies on an unusual mechanism mediated by a protein complex formed by Ran and NTF2. Additionally, Chickadee (Chic) was shown to function as a negative regulator of IRP1A nuclear import. These findings provided molecular evidence for how IRP1A nucleocytoplasmic transport occurs in Drosophila melanogaster. RanBP3 was also characterized as a novel regulator of intracellular iron trafficking, which is critical for transporting cytosolic iron into the mitochondrion for heme biosynthesis and iron-sulfur cluster biogenesis.<br>
The other gene that I investigated affects ecdysone biosynthesis via a different mechanism than RanBP3. Suppressor of variegation 2-10 or Su(var)2-10 encodes a protein inhibitor of activated STATs (PIAS) protein that regulates chromosome structure and function. Losing Su(var)2-10 function in the prothoracic gland caused lethality and an overgrowth of the tissue, which suggests a feedback control mechanism to produce more ecdysone to sustain normal larval-to-pupal transition. I show that Su(var)2-10 is required for the transcriptional regulation of neverland (nvd), which encodes an enzyme required for the conversion of dietary cholesterol to 7-dehydrocholesterol (7dC). Prior to this work, two transcription factors (TFs) were known to cooperatively regulate the nvd gene, Séance and Molting defective (Mld). Only the expression of séance, but not that of mld, was affected by Su(var)2-10 loss-of-function in the PG cells. This finding established a regulatory network in which nvd transcription is controlled by a protein that functions upstream of a known transcription factor. Mass spectrometry analysis identified Histone H2A (His2A) as a Su(var)2-10-interacting protein. Knocking down His2A in the prothoracic gland down-regulated ecdysone biosynthetic gene transcription, suggesting that Su(var)2-10 may regulate nvd transcription via histone modification, presumably SUMOylation.</p>