Confirmation and characterization of IMD immune-responsive enhancers in

Drosophila melanogaster using flow cytometry

Caitlin Sauer, Lianne B. Cohen, Zeba Wunderlich
Department of Biology, Boston University

Abstract Methods Conclusion/Future Directions

The immune system of Drosophila melanogaster is a simple • Enhancers with minimal GFP signal show low to no
yet effective model for studying fundamental principles of Unique Enhancer
Sequence Electroporate activation when the IMD pathway is stimulated
Enhancer-Reporter
immunity. Recently, a STARR-seq experiment in D. into S2* cells • Need alternative information to confirm enhancer
melanogaster identified IMD responsive enhancers, key to activity in the IMD pathway
immune regulation. To validate and characterize these Ampicillin
Resistance Gene
DSC Promoter • Confirmed known enhancers for PGRP and Mtk to
findings, we cloned 10 enhancer sequences upstream of GFP validate findings in the STARR-seq experiment
(green fluorescent protein) and transfected D. melanogaster • Percentage of GFP positive cells are correlated with the
Origin of Fluorescence Activated
S2* cells. Enhancer sequences were selected based on their Replication
EGFP Cell Analyzer number of Relish-binding sites
proximity to immune genes, transcription factor binding site • Cells are individually analyzed according to
• Future experiments include a more robust study design,
density, sequence length, and STARR-seq expression levels. their fluorescence intensity using lasers implementing transfection control plasmids
• Processed through gradient of fluorescence
After immune activation of cells, GFP fluorescence levels emission filters miRFP 670
hr5-pIEX-miRFP670-IE1 terminator
were quantified using Fluorescence-Activated Cell Sorting • Precise detection of wavelength and
fluorescence strength translated into histograms
(FACS), allowing for the precise measurement of the IE1
Constitutively active promoter – cells
Relish is an NF-kB Promoter transfected successfully will continuously
enhancer-driven transcriptional activity in response to immune Associated Gene
Chosen Enhancer Sequences
Reason for Studying transcription factor express red protein = red fluorescence
signaling. 7 of the 10 enhancers showed increased GFP signal, PGRP Known immune enhancer specifically required for IE1 Terminator
CrebA Gene downstream of the immune pathways the induction of the IMD
indicating their functional role in the IMD response, while the Salm High activity score, immune specific, associated with immune response in D.
*Expected FACS
histogram
developmental genes
remaining 3 showed no significant activity, suggesting limited Lectin High activity score (strong signal when sequenced)
melanogaster hr5 Enhancer

involvement or the need for further validation. Our results Daisho

Aqz
Required for defense against fungal pathogens
6 Relish sites

provide insights into the functional diversity of enhancer Rdo

Limpet
5 Relish sites
1 Relish site
Enhancers with a
different amount of
elements in the immune response of D. melanogaster, Fas3 High activity score (strong signal when sequenced)
Relish transcription
Mtk Known immune enhancer
highlighting potential new regulatory sequences that may factor binding sites
10 enhancers and their closest proximal Plasmids are co-transfected into the same
contribute to immune pathway modulation. gene, and reasoning for being chosen cells, ensuring that cells expressing miRFP- During analysis, cells expressing
670 also contain the GFP enhancer reporter, miRFP-670 are gated off using
allowing the transfection control to identify flow-cytometry histograms to
only the successfully transfected cells isolate transfected cells

Introduction • Allows for the standardization of different enhancers

Results/Graphs • Further experiments include stimulating the Toll pathway
• Experiments will include inducing IMD and Toll
Uninduced individually, as well as inducing both at the same time
CrebA Induced
Events

GFP
Histogram of STARR-seq enhancer closest in proximity to the gene, CrebA
Uninduced Sample: 37.7% GFP positive with a Geometric Mean of 8.19
Induced Sample: 73.3% GFP positive with a Geometric Mean of 38.68
• Does the activation of one pathway influence the other
Salm during dual-induction?
• Same or different response from both Toll and IMD
Events

• Is there cross-regulation between the Toll and IMD

pathways at the enhancer level?
GFP • Are some IMD enhancers found in the STARR-seq
Histogram of STARR-seq enhancer closest in proximity to the gene,
• Innate immune response in D. melanogaster, involves two Salm experiment active in Toll as well?
immune pathways – Toll and IMD – work together to fight Uninduced Sample: 0.1% GFP positive with a Geometric Mean of 6.82 • Are certain enhancers more responsive to one pathway over
Induced Sample: 0.9% GFP positive with a Geometric Mean of 5.07
infectious microorganisms the other?
PGRP
• Transcription factors facilitate DNA transcription
• Enhancers, as cis-regulatory sequences, act as transcription
Events

factor binding sites, aiding in the regulation of gene

expression independent of location and proximity Acknowledgements
• Variations in cis-regulatory elements can lead to functional GFP
Histogram of STARR-seq enhancer closest in proximity to the gene, PGRP
changes, impacting key biological pathways in humans and Uninduced Sample: 1.5% GFP positive with a Geometric Mean of 5.34 I would like to thank the Wunderlich Lab for their support and
flies Induced Sample: 24.7% GFP positive with a Geometric Mean of 6.51
guidance throughout this project. Special thanks to my mentor,
Lianne Cohen, for her invaluable mentorship, and to Zeba
STARR-seq Protocol
Wunderlich for her leadership and insightful feedback. This
work would not have been possible without their expertise and
encouragement.

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