Stephen S. Gisselbrecht, Luis A. Barrera, Martin Porsch, Anton Aboukhalil, Preston W. Estep III et al.
Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these elements in development, responses to environmental stresses, and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here, we have developed ‘enhancer-FACS-Seq’ (eFS) technology for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS to be active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them.
Stephen S. Gisselbrecht, Luis A. Barrera, Martin Porsch, Anton Aboukhalil, Preston W. Estep III, Anastasia Vedenko, Alexandre Palagi, Yongsok Kim, Xianmin Zhu, Brian W. Busser, Caitlin E. Gamble, Antonina Iagovitina, Aditi Singhania, Alan M. Michelson, Martha L. Bulyk. Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos. 2013-07-14. https://doi.org/10.1038/nmeth.2558
The second injection batch of the cCRM library was assayed in stage 11–12 embryos carrying the duf:CD2 muscle-founder marker, and the rare GFP-positive CD2-positive population was pooled before sequencing. The pooled target was compared with five size-matched duf:CD2 input-control samples.
The pooled cCRM library was assayed in stage 11–12 Drosophila embryos carrying the twi:CD2 mesoderm marker, and GFP-positive CD2-positive cells were collected. Activity was quantified against matched twi:CD2+ input cells in three target and three input replicates.
The pooled cCRM library was assayed in stage 11–12 Drosophila embryos carrying the twi:CD2 mesoderm marker, and GFP-positive CD2-negative cells were collected. Activity was quantified against matched twi:CD2− input cells in three target and three input replicates.
The cCRM library was assayed in stage 11–12 embryos carrying the Mef2-I-E_D5:CD2 marker, and the rare GFP-positive CD2-positive fusion-competent myoblast population was pooled before sequencing. The pooled target was compared with five size-matched Mef2-I-E_D5:CD2− input/variance-control samples.
The cCRM library was assayed in stage 11–12 embryos carrying the Mef2-I-E_D5:CD2 marker, and GFP-positive CD2-negative cells were collected as an approximately whole-embryo control population. Activity was quantified against ordinary Mef2-I-E_D5:CD2− input cells in three target and three input replicates.
The second injection batch of the cCRM library was assayed in stage 11–12 embryos carrying the duf:CD2 muscle-founder marker, and GFP-positive CD2-negative cells were collected as an approximately whole-embryo control population. Activity was quantified against ordinary duf:CD2− input cells in six target and six input replicates.
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 54 files (ZIP)E-GEOD-41503.idf.txtE-GEOD-41503.sdrf.txtgeo_processed/GSM1018361_lib36TG_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018362_lib39MA_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018363_lib40MG_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018364_lib41TA_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018365_lib44MB_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018366_lib45MH_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018367_lib46TB_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018368_lib47TH_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018369_lib49MC_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018370_lib50MI_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018371_lib51TC_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018372_lib52TI_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018373_lib54MD_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018374_lib55MJ_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018375_lib56TD_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018376_lib57TJ_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018377_lib60MK_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018378_lib61TE_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018379_lib62TK_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018380_lib65ME_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018381_lib66TF_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018382_lib67TL_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018383_lib70MF_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1018384_lib71ML_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138197_lib73dufN_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138198_lib74dufQ_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138199_lib75dufY_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138200_lib78dufO_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138201_lib79dufR_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138202_lib82dufP_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138203_lib83dufZ_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138204_lib86dufS_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138205_lib87dufW_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138206_lib88dufAA_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138207_lib91dufT_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138208_lib92dufX_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138209_lib96dufU_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138210_lib98dufEE_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138211_lib100dufM_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138212_lib101dufV_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138213_lib102dufDD_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtgeo_processed/GSM1138214_lib103dufFF_segemehl_H0_M100_E5_D2_A80_cCRMmatched_cCRMs.txtSOURCE_MANIFEST.txtSupplementary_Table_1_cCRM_library.xlsSupplementary_Table_2_cell_counts.xlsSupplementary_Table_3_sequencing_QC.xlsSupplementary_Table_4_eFS_results.xlsSupplementary_Table_5_validation_and_adjacent_genes.xlsSupplementary_Table_6_validation_summary.xlsSupplementary_Table_7_motif_results.xlsSupplementary_Table_8_feature_summary.xlsSupplementary_Text_and_Figures.pdf