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D. Melanogaster Synaptic Gene Motifs in Embryonic Neurons between 10-12 hours

Tasawwar E. Rahman1,2, James Kentro3, Erica Larschan3, Kate O’Connor-Giles4

1Program in Liberal Medical Education, The Warren Alpert Medical School of Brown University, 2Center for Computational and Molecular Biology,

3Department of Molecular Biology, Cell Biology, and Biochemistry, 4Department of Neuroscience, Brown University, Providence, RI 02912

Introduction

Synapses form the basis on which neurons communicate. Synaptogenesis is a critical component of neuronal development and requires that hundreds of genes be simultaneously expressed. Yet the mechanism behind synaptic gene coordination remains poorly characterized.

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The O’Connor-Giles lab and others have previously identified a temporal pattern of expression in which pan-neuronal and subtype-specific synaptic genes show increased expression corresponding to periods of heightened synaptogenesis.1 To better understand this coordination, we used DNA-seq data from to Furlong Lab to identify several transcription factor binding motifs unique to neurons at 10 to 12 hours in embryonic development.

Discussion

  • Our results create a model of which transcription factors may turn on associated genes in the 10 to 12 hour time points where we see significant synaptic activity.
  • Identifying these gene/TFs allow us to perform further experimentation to understand the coordination mechanism behind the temporal activation of synaptic genes.
  • Of our 4 pioneer transcription factors of interest– DEAF1, CLAMP, L(3)neo38, and GAF– we found binding motifs in the 10 to 12 hour time points for GAF, CLAMP (via competition with GAF for the Trl binding motif), and DEAF1. When subsetting for synaptic genes, we didn’t find any binding sites for these TFs.

Peaks

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  • Top band are peaks expressed only in neurons between 10 to 12 hours. We found 4245 peaks in 10 to 12 only vs 19467 without subtracting for other tissue/time points.
  • The 2nd band are peaks in neurons between 10 to 12 hours within 3k bps up or down stream (~1 gene) of synaptic genes.
  • The 3rd band is the same but instead 10k bps up or down stream.

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Motifs

  • 18 binding motifs found for peaks only in neurons between 10 to 12 hours.

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  • 37 binding motifs found for peaks within 3k bps of synaptic genes in neurons between 10 to 12 hours.

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Future Directions

  • In order to better understand the relationship between individual gene expression and the coordination of synaptic genes, we need to perform knock out or knock down experiments on our genes of interest.
  • I will be continuing my work in the Larschan and O’Connor-Giles labs learning how to dissect larval brains for RNA and ATAC-seq as well as learning to dissect and characterize larval neuromuscular junctions.
  • We will be expanding our computational focus to look at single cell data.

Methodology

To analyze our data, we used DNA-seq data from the Furlong Lab and modified a 21 step NGS sequencing pipeline found in a paper by Reddington et al.2 To preprocess our data, we trimmed the sequences using Trimgalore, aligned them to the reference genome (dm3) using Bowtie2, filter/sorted and merged technical replicates using Samtools, removed duplicates using Picard, and created pseudo replicates of the individual biological replicates and the merged biological replicates using Samtools. We then peak called using MACS2, ran IDR analysis using IDR, analyzed consistency between IDR sets, subtracted other tissue/time-point types from the neuronal peaks at 10 to 12, and windowed for synaptic genes all using Bedtools. Finally, we ran motif analysis in HOMER.

1Kurmangaliyev, Y. Z., Yoo, J., Valdes-Aleman, J., Sanfilippo, P., & Zipursky, S. L. (2020). Transcriptional Programs of Circuit Assembly in the Drosophila Visual System. Neuron, 108(6), 1045–1057.e6. https://doi.org/10.1016/j.neuron.2020.10.006

2Reddington, J. P., Garfield, D. A., Sigalova, O. M., Karabacak Calviello, A., Marco-Ferreres, R., Girardot, C., Viales, R. R., Degner, J. F., Ohler, U., & Furlong, E. E. M. (2020). Lineage-Resolved Enhancer and Promoter Usage during a Time Course of Embryogenesis. Developmental cell, 55(5), 648–664.e9. https://doi.org/10.1016/j.devcel.2020.10.009

Acknowledgements

I want to thank my mentor James Kentro for all his support and guidance throughout this process. I also want to thank Drs. Larschan and O’Connor-Giles for welcoming me into their labs this summer and the PLME without whose funding funding this research would not be possible.