Crosstalk between vrille transcripts, proteins and regulatory elements controlling circadian rhythms and development in Drosophila
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ABSTRACT:
PROVIDER: PRJNA647453 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12274600.fastq.gz | Fastqsanger.gz | |||
| SRR12274601.fastq.gz | Fastqsanger.gz | |||
| SRR12274602.fastq.gz | Fastqsanger.gz | |||
| SRR12274603.fastq.gz | Fastqsanger.gz | |||
| SRR12274604.fastq.gz | Fastqsanger.gz |
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Project description:The vrille (vri) gene encodes a bZIP transcriptional repressor that is required for Drosophila patterning and cell proliferation during development as well as circadian behavior in adults. Three alternate first exons produce five different vri transcripts, where first exons 1a and 1b initiate two transcripts during development that produce the 610aa short VRI isoform and first exon 1c initiates three transcripts in adults that produce the 729aa long VRI isoform. To test whether long VRI is necessary for circadian clock function, we generated a mutant (vriΔ679) that eliminates transcripts encoding long VRI. The vriΔ679 mutant is viable, confirming that developmental functions of vri are driven by transcripts from exons 1a/1b, yet behavioral rhythms persist in vriΔ679 flies, demonstrating that vri transcripts encoding short VRI are sufficient for clock output. E-box regulatory elements upstream of exon 1c that drive rhythmic transcription in adults are also required for development, implying that these E-boxes also drive vri transcription from exons 1a/1b. Surprisingly, there is almost no long VRI present in adults even though the vast majority of transcripts encode long VRI, apparently because the ATG start codon for long VRI has a poor Kozak sequence context. Consequently, short VRI is the predominant isoform in adults and is sufficient for clock output. We conclude that vri-ADF transcripts driven by E-boxes upstream of exon 1c primarily control circadian rhythms by producing short VRI , whereas vri-E transcripts that are also regulated by E-boxes upstream of exon 1c mediate developmental patterning/cell proliferation through short VRI.
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Project description:The vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vri Δ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vri Δ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.
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Project description:Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.
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Project description:Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e. distal convoluted tubule (DCT) and connecting tubule (CNT) and, the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, M-oM-^AM-!ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function. Experiment Overall Design: We examined the temporal profiles of gene expression in mouse distal nephron segments and collecting ducts. The RNA was extracted from microdissected distal convoluted tubules and connecting tubules (DCT/CNT samples) or, cortical collecting ducts (CCD samples). Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT M-bM-^@M-^S Zeitgeber (circadian) time, indicates time of light-on as ZT0 and time of light-off as ZT12). The microarray hybridization was performed in duplicates on two pools of RNA composed of equivalent amounts of RNA prepared from five animals at each ZT time-point.
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