Identification of a Hormone-Responsive Enhancer Element in the Cytochrome b561 gene in Mouse Hippocampal Neurons

Presentation Number: SUN 264
Date of Presentation: April 2nd, 2017

Jose Ezekiel Clemente Espina* and Pia Dano Bagamasbad
University of the Philippines Diliman, Quezon City, Philippines

Abstract

Thyroid hormones (TH) and glucocorticoids (GC) influence neuronal development through their action as ligand-activated transcription factors. Though studies have elucidated the effects of TH on early brain development and the context-dependent effects of GC on learning and memory formation, little is known about the combined effects of both hormones in the brain, and consequently neuronal morphology and function. Microarray studies have found that the Cytochrome b561 (Cyb561) gene was the most robustly and synergistically upregulated gene in mouse hippocampal neurons in response to TH and GC treatment. The Cyb561 gene encodes an electron transport protein which recycles ascorbate for use as a cofactor in the alpha-amidation and activation of neuropeptides. Mutations in a Cyb561 gene orthologue in Drosophila have been found to be associated with deficits in learning and memory formation suggesting an important role for Cyb561 gene in neuronal function. To determine the molecular mechanism of synergy of TH and GC action on Cyb561 gene regulation in the brain, mouse hippocampal neurons (HT22 cells) were treated with TH, GC, and TH plus GC, followed by total RNA extraction, reverse transcription (RT), and pre-mRNA and mRNA expression analysis using SYBR Green quantitative PCR (qPCR). Hormone treatments were also done in the presence of a protein synthesis inhibitor cycloheximide to determine if the Cyb561 gene is a direct target of TH receptor (TR) and GC receptor (GR) action. Gene expression analysis showed that Cyb561 mRNA is directly and synergistically upregulated by TR and GR, which exert their effects through an increase in transcriptional rate. Using in silico analysis from publicly available ENCODE data, we identified a conserved 320 bp enhancer region containing a candidate GC response element (GRE) located 9.4 kb upstream of the Cyb561 transcription start site. Enhancer-reporter assays of the candidate enhancer element showed that it was responsive to GC but not TH. Transcript expression analysis showed the identified Cyb561 enhancer was being transcribed into an enhancer RNA in the same pattern as Cyb561 induction by TH and GC, indicating that the identified enhancer may have been incomplete. A larger 850 bp putative enhancer region containing both GRE and TH response element (TRE) was cloned into pGL4.23 for further testing. Taken together, we were able to show that the Cyb561 gene is synergistically regulated by TH and GC through direct targeting by TR and GR, and we identified a hormone-responsive enhancer element 9.4 kb upstream of Cyb561 transcription start site which was also being transcribed into an enhancer RNA that is induced synergistically by TH and GC. Our findings support the hypothesis that the Cyb561 gene may serve as an important mediator in integrating the effects of the two neuroendocrine axes in regulating neuronal processes related to development, and learning and memory.

 

Nothing to Disclose: JECE, PDB