Regulatory Mechanisms of Metamorphic Neuronal Remodeling Revealed Through a Genome-Wide Modifier Screen in Drosophila melanogaster

Abstract Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

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VPS37A directs ESCRT recruitment for phagophore closure

The Journal of Cell Biology ◽

10.1083/jcb.201902170 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3336-3354 ◽

Cited By ~ 17

Author(s):

Yoshinori Takahashi ◽

Xinwen Liang ◽

Tatsuya Hattori ◽

Zhenyuan Tang ◽

Haiyan He ◽

...

Keyword(s):

Mammalian Cells ◽

Growth Factor Receptor ◽

Multivesicular Body ◽

Regulatory Mechanisms ◽

Endosomal Sorting ◽

Aaa Atpase ◽

Escrt Machinery ◽

Genome Wide ◽

A Genome ◽

Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic

eLife ◽

10.7554/elife.16096 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 29

Author(s):

Joel M Swenson ◽

Serafin U Colmenares ◽

Amy R Strom ◽

Sylvain V Costes ◽

Gary H Karpen

Keyword(s):

Cell Cycle ◽

Image Analysis ◽

Drosophila Melanogaster ◽

Gene Silencing ◽

Epigenetic Factors ◽

Heterochromatin Protein ◽

Genome Wide ◽

A Genome ◽

Drosophila Heterochromatin ◽

Heterochromatin Domain

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

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