Epigenetic regulation of ecdysone receptor B1 (EcR-B1) in the Drosophila melanogaster brain

Emily Jane Sterner

doi:10.17918/00011178

The ecdysone receptor B1 isoform (EcR-B1) is required for response to developmental pulses of the molting hormone ecdysone, leading to changes in gene expression responsible for neuronal remodeling in the Drosophila brain during pupation. Expression of EcR-B1 is regulated, in part, by epigenetic factors which can edit or read the epigenetic landscape at cis-regulator regions. The reader domain-containing protein Kismet (Kis) is known to be required for proper EcR-B1 expression. While the mechanism by which it promotes EcR-B1 expression is poorly understood, it is known to promote epigenetic marks at EcR promoter regions associated with active gene expression, including acetylation at H4K16. Removal of this mark would be associated with a closed chromatin conformation, decreasing transcription of ecr-b1 mRNA. Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl marks. General inhibition of HDACs using the inhibitor SAHA was previously found to rescue the reduced EcR-B1 protein and mRNA levels associated with loss or knockdown of Kis; SAHA also rescued phenotypes associated with reduced EcR-B1, such as axon pruning failure and defective immediate recall memory. Thus, we hypothesized that EcR is a common target of both Kis and HDACs. However, the specific HDACs involved in the epigenetic regulation of EcR-B1 were not known. The aim of my thesis work was to determine which HDACs were important for control of EcR-B1 expression in the Drosophila brain; my top candidates for this role were the two class I HDACs in Drosophila, HDAC1 (Rpd3) and HDAC3, because they are known to remove H4K16ac. Previous work in the lab also showed HDAC inhibitors more specific to class I HDAC performing well in rescuing the effects of Kis knockdown. When I tested the effects of HDAC1 or HDAC3 RNAi in fly brains also containing a Kis RNAi, I found that knockdown of either HDAC1 nor HDAC3 was not sufficient to rescue EcR-B1 mRNA or protein levels. I next treated flies with either the ketone body [beta]-hydroxybutyric acid ([beta]OHB) or the fatty acid sodium butyrate (SB), both known class I and II HDAC inhibitors. I found that both of these commonly-found metabolites were capable of rescuing EcR-B1 protein and mRNA levels in a Kis knockdown background. However, though [beta]OHB and SB are thought to have the same mechanism of action due to their similar structures, I found differences in how they affected the system. SB was capable of rescuing EcR-B1 protein and mRNA levels without affecting Kis levels, a result which mirrored previous results with SAHA. [beta]OHB increased both ecr-b1 and kis mRNA levels in one of the Kis knockdown conditions, implying that it may work to rescue ecr-b1 mRNA levels by increasing kis mRNA levels. However, [beta]OHB was also capable of rescuing EcR-B1 protein levels in the absence of a functional Kis protein. These results imply a potential dual role for [beta]OHB in this system, as well as differences between the mechanisms of action for [beta]OHB and SB. Taken together, this work shows a role for class I HDACs in the epigenetic regulation of EcR-B1, in addition to new discoveries in the roles of key metabolites known to affect epigenetic factors.

Epigenetic regulation of ecdysone receptor B1 (EcR-B1) in the Drosophila melanogaster brain

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Epigenetic regulation of ecdysone receptor B1 (EcR-B1) in the Drosophila melanogaster brain

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