Vincent Loubiere, Bernardo P. de Almeida, Michaela Pagani, Alexander Stark
Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and about a million enhancer pairs toward housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are super-additive and combine multiplicatively. Super-additivity between developmental enhancers is promiscuous and neither depends on the enhancers’ endogenous genomic contexts nor on specific transcription factor motif signatures. However, it can be further boosted by Twist and Trl motifs and saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene regulation in complex multi-enhancer developmental loci and genomically clustered housekeeping genes, providing a rationale to interpret the transcriptional impact of non-coding mutations at different loci.
Vincent Loubiere, Bernardo P. de Almeida, Michaela Pagani, Alexander Stark. Developmental and housekeeping transcriptional programs display distinct modes of enhancer-enhancer cooperativity in Drosophila. 2024-10-03. https://doi.org/10.1038/s41467-024-52921-2
A wild-type/control enhancer-pair library was assayed with an approximately 2-kb inert genomic spacer downstream of DSCP in untreated S2 cells to test whether increased enhancer separation preserves cooperativity. The deposited table contains 38,898 oriented pair measurements.
The 1000-oligo wild-type/control pair library was assayed toward DSCP in Drosophila ovarian somatic cells (OSC), including OSC-specific candidate sequences. The deposited table contains 716,384 oriented pair measurements.
A focused 165-sequence library containing housekeeping, developmental, shared, and control oligos was paired around a 300-bp spacer and assayed toward the housekeeping RpS12 core promoter in untreated S2 cells. The deposited table contains 28,031 oriented pair measurements.
An engineered oligo library containing wild-type enhancer sequences and motif perturbations was combinatorially paired around a 300-bp spacer and assayed toward DSCP in untreated S2 cells. The library perturbs Twist, Trl/GAGA, and Dref motif content and the deposited table contains 620,742 oriented pair measurements.
The 1000-oligo wild-type/control pair library was assayed toward DSCP in S2 cells treated with 20-hydroxyecdysone after electroporation. The deposited table contains 716,152 oriented pair measurements and includes ecdysone-inducible candidate sequences.
A 1000-oligo wild-type/control library was combinatorially fused around a 300-bp inert spacer and assayed toward the developmental synthetic core promoter (DSCP) in untreated S2 cells. The deposited table contains 715,479 oriented pair measurements.
The focused 165-sequence housekeeping/developmental/shared/control library was paired around a 300-bp spacer and assayed toward the developmental synthetic core promoter (DSCP) in untreated S2 cells. The deposited table contains 27,536 oriented pair measurements.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 16 files (ZIP)Focused_oligo_pool_sequences.tsvGSE245033_filelist.txtGSE245033_Focused_oligo_pool_dCP_S2_FC_table.txt.gzGSE245033_Focused_oligo_pool_hkCP_S2_FC_table.txt.gzGSE245033_Mutated_oligo_pool_dCP_S2_FC_table.txt.gzGSE245033_WT_2kb_spacer_dCP_S2_FC_table.txt.gzGSE245033_WT_oligo_pool_dCP_OSC_FC_table.txt.gzGSE245033_WT_oligo_pool_dCP_S2_ecdysone_FC_table.txt.gzGSE245033_WT_oligo_pool_dCP_S2_FC_table.txt.gzMutated_oligo_pool_sequences.tsvOligo_pool_summary.tsvPCR_primers.tsvPWM_list.tsvREADME.txtSource_Data.xlsxWT_oligo_pool_sequences.tsv