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Drosophila L3 ATAC-seq at 24h APF

ABSTRACT: The layered compartmentalization of functionally-related synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila melanogaster visual system is comprised of 10 discrete layers (M1-M10), within which neural computations underlying distinct visual features are processed. As such, the Drosophila medulla neuropil serves as a model system for understanding layered compartmentalization of synaptic connectivity. The first step leading to the establishment of neurite layer specificity in the outer medulla (M1-M6) is the innervation of one of two broad, primordial domains that will subsequently expand and transform into the M1-M6 layers. We previously found that the transcription factor dFezf cell-autonomously directs L3 neurons to their proper early broad domain before they innervate and form synapses specifically within the M3 layer of the mature medulla. Here, we examined the transcriptomes of wild type and dFezf mutant L3 neurons by RNA-seq and identified downstream target genes that are regulated by dFezf. We show that dFezf controls L3 layer specificity through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). During the period of broad domain selection, dFezf limits slp1 expression in L3 neurons. Slp1 is upregulated in dFezf-null L3 neurons, and ablation of slp1 fully rescues the targeting defect observed in dFezf-null L3 growth cones. Surprisingly, L3 innervation of the M3 layer after broad domain selection requires early slp1 expression. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation. Moreover, we performed ATAC-seq in wild type L3 neurons and identified the accessible regions in the L3 genome, where dFezf may directly bind and regulate gene expression.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE163307 | GEO | 2020/12/17

REPOSITORIES: GEO

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Project description:The layered compartmentalization of functionally-related synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila melanogaster visual system is comprised of 10 discrete layers (M1-M10), within which neural computations underlying distinct visual features are processed. As such, the Drosophila medulla neuropil serves as a model system for understanding layered compartmentalization of synaptic connectivity. The first step leading to the establishment of neurite layer specificity in the outer medulla (M1-M6) is the innervation of one of two broad, primordial domains that will subsequently expand and transform into the M1-M6 layers. We previously found that the transcription factor dFezf cell-autonomously directs L3 neurons to their proper early broad domain before they innervate and form synapses specifically within the M3 layer of the mature medulla. Here, we examined the transcriptomes of wild type and dFezf mutant L3 neurons by RNA-seq and identified downstream target genes that are regulated by dFezf. We show that dFezf controls L3 layer specificity through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). During the period of broad domain selection, dFezf limits slp1 expression in L3 neurons. Slp1 is upregulated in dFezf-null L3 neurons, and ablation of slp1 fully rescues the targeting defect observed in dFezf-null L3 growth cones. Surprisingly, L3 innervation of the M3 layer after broad domain selection requires early slp1 expression. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation. Moreover, we performed ATAC-seq in wild type L3 neurons and identified the accessible regions in the L3 genome, where dFezf may directly bind and regulate gene expression.

Project description:HSF1 binds DNA via the DBD domain, causing gene upregulation during HS. We assessed the effect of RD-mediated phase separation on chromatin targeting of HSF1 using Cut&Tag followed by high-throughput sequencing to map genome-wide binding of LLPS-competent versus LLPS-incompetent HSF1 mutants under both HS and NHS conditions. Comparing with WT HSF1 under NHS, both WT under HS and M1 under NHS showed increased and broad binding to enhancers and distal intergenic region, with binding most enriched in expected motifs of HSF-related transcription factors. To further assess the role for IDR-induced LLPS in chromatin targeting of HSF1, we used several additional strategies. First, the treatment of 1,6-hexanediol markedly decreased chromatin occupancy both of WT under HS and M1 under NHS conditions. Second, we interrogate chromatin binding of LLPS-deficient mutant M3. Cut&Tag analysis revealed that M3 showed decreased chromatin binding compared to WT under HS and M1 under NHS. The decreased chromatin binding of M3 to chromatin was not due to the loss of its DNA binding ability, as the EMSA assay revealed that M3 was still capable of binding HSE. Instead, the decreased chromatin binding reflects the loss of inter-molecular interaction between HSF1 that holds LLPS. Furthermore, M3 in heat shocked cells shows similar reduced genomic targeting and shallow binding pattern as NHS cells . Third, the enrichment of transcriptional apparatus RNA Pol II, CYCT1, BRD4 to HSF1 target genes also depend on whether HSF1 can phase separate at these sites. Lastly, we conducted live cell single molecule imaging to evaluate chromatin binding kinetics of LLPS-deficient mutant M3 relative to WT HSF1. Measurements of single molecule displacement and diffusion coefficient showed M3 to be significantly more mobile than WT under HS, which suggests M3 was less confined within phase-separated puncta compared with LLPS-competent HSF1. Consistent with this result, super resolution imaging of M3 also showed decreased cluster formation at HSP gene foci but maintained nSBs formation. Altogether, LLPS-forming capability of HSF1 is essential for the efficient recruitment of HSF1 and transcriptional apparatus to HSP gene loci.

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Drosophila L3 ATAC-seq at 24h APF

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