Experiment / E3OR8YKMESilencer / Repressor MPRA

RD-seq repressive-domain screen with the zfh1-DSCP reporter

Systematic identification and characterization of repressive domains in Drosophila transcription factors

A pooled library of 209,495 synthetic 150-bp DNA fragments encoding 50-amino-acid tiles from 1,133 Drosophila transcription-related genes was fused to Gal4-DBD and tested in Schneider S2 cells carrying an integrated UAS-EGFP reporter driven by the zfh1 enhancer and Drosophila synthetic core promoter. Two biological replicates were separated into GFP-negative and GFP-positive fractions by FACS three days after transfection and quantified by UMI-aware paired-end sequencing; the table contains the 131 RD regions called in both zfh1 replicates.

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Organism: Fruit fly
Taxonomy ID: NCBITaxon:7227
Biosample: CVCL:Z232
Reference genome: Not reported / not applicable
Design focus: Other
Region of interest: Not reported / not applicable

Perturbation & assay details

Gal4-DBD candidate-library transfection; FACS separation into GFP-negative and GFP-positive fractions 3 days later

RD-seq is a pooled protein-effector-domain reporter screen adapted from tAD-seq rather than a conventional cis-regulatory-element MPRA. Each 150-bp oligo encoded a Drosophila protein tile, was expressed as a Gal4-DBD fusion, and was tethered to 14 UAS sites in a CRISPR-integrated reporter at chr2L:9094918. GFP-negative and GFP-positive fractions were sequenced after three days; mapped fragments were collapsed by oligo coordinates and 10-bp UMIs, and repression was quantified as enrichment in GFP-negative over GFP-positive coverage.

Processed data

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Column dictionary · 43 definitions

element_id: Generated stable identifier combining FlyBase gene ID, transcript ID, and the author RD name.
gene_id: FlyBase gene identifier (FBgn) for the protein-coding locus.
transcript_id: FlyBase transcript identifier (FBtr) used for the tiled coding sequence.
gene_symbol: Drosophila gene symbol mapped from the EV1 candidate-gene manifest.
rd_name: Author-provided name for the repressive domain region.
reporter: Reporter condition for this experiment: zfh1.
rd_start_nt: Author EV3 start of the RD region in nucleotides relative to the full-length transcript/protein coding sequence; not a genomic coordinate.
rd_end_nt: Author EV3 end of the RD region in nucleotides relative to the full-length transcript/protein coding sequence; not a genomic coordinate.
rd_width_nt: Author EV3 width of the extended RD region in nucleotides.
rd_length_aa: Length of the extended RD region in amino acids.
rd_nt_sequence: Nucleotide sequence of the extended RD region from EV3.
rd_aa_sequence: Amino-acid sequence of the extended RD region from EV3.
best_oligo_id: Author ID of the highest-scoring 150-bp candidate oligo within the RD region.
best_oligo_position_nt: Author-reported transcript/CDS-relative position string for the highest-scoring oligo.
best_oligo_nt_sequence: Nucleotide sequence of the highest-scoring 150-bp oligo.
best_oligo_aa_sequence: Amino-acid sequence encoded by the highest-scoring oligo.
best_oligo_log2fc: Author log2 enrichment score for the best oligo in the zfh1 reporter.
best_oligo_pvalue: Author hypergeometric P-value for the best oligo in the zfh1 reporter.
best_oligo_padj: Author Benjamini-Hochberg adjusted P-value for the best oligo in the zfh1 reporter.
replicate_1_sample: GEO sample accession for zfh1 biological replicate 1.
replicate_1_call_width_nt: Width of the matching RD call in zfh1 replicate 1 from the GEO RD_calls file.
replicate_1_cov_neg_fragments: Reported fragment coverage in the GFP-negative fraction for zfh1 replicate 1.
replicate_1_cov_pos_fragments: Reported fragment coverage in the GFP-positive fraction for zfh1 replicate 1.
replicate_1_log2fc: Reported RD-call log2 enrichment (GFP-negative over GFP-positive) for zfh1 replicate 1.
replicate_2_sample: GEO sample accession for zfh1 biological replicate 2.
replicate_2_call_width_nt: Width of the matching RD call in zfh1 replicate 2 from the GEO RD_calls file.
replicate_2_cov_neg_fragments: Reported fragment coverage in the GFP-negative fraction for zfh1 replicate 2.
replicate_2_cov_pos_fragments: Reported fragment coverage in the GFP-positive fraction for zfh1 replicate 2.
replicate_2_log2fc: Reported RD-call log2 enrichment (GFP-negative over GFP-positive) for zfh1 replicate 2.
mean_rd_log2fc: Author EV3 mean RD-region log2FC across the two zfh1 replicates.
other_reporter_mean_rd_log2fc: Author EV3 mean RD-region log2FC across the two ent1 replicates, included for cross-reporter context.
mean_activity_both_reporters: Author EV3 mean of the zfh1 and ent1 RD-region mean log2FC values.
rd_log2fc_preference_ent1_minus_zfh1: Author EV3 difference between ent1 and zfh1 mean RD-region log2FC.
reporter_preference_class: Author EV3 reporter preference classification using a 1.3-fold threshold: ent1, zfh1, or global.
stampfel_2015_cluster: Cluster number from the Stampfel et al. 2015 transcription-factor activity comparison, when available.
stampfel_2015_sum: Sum of luciferase signals across 24 Stampfel et al. 2015 contexts, when available.
stampfel_2015_repressor: Author categorization of the full-length protein as a Stampfel repressor.
known_or_literature_repressor: Author annotation of whether the full-length protein is a known/literature repressor.
rd_known: Author annotation of whether the RD has been described previously.
literature_reference: Literature citation supporting repressor status, when available.
literature_reference_doi: DOI for the repressor-status literature citation, when available.
rd_reference: Literature citation for a previously described RD, when available.
rd_reference_doi: DOI for the previously described RD citation, when available.

Quality control

The authors collapsed mapped fragments by oligo coordinates and near-matching UMIs, calculated hypergeometric enrichment and Benjamini-Hochberg FDR, and called RD regions using at least 10 fragments in each GFP fraction, enrichment of at least 1.5-fold, hypergeometric P <= 1e-5 across at least 60 bp, with flanking CDS extension until P > 1e-3. For each reporter, only regions overlapping at least 50% between the two biological replicates were retained. The processed table contains only Dataset EV3 rows with presence.RD.region.zfh1=1, giving 131 reproducible zfh1 regions; no additional activity threshold was imposed.

Curation notes

This is an MPRA-style repressor-domain screen: it measures trans-acting protein fragments with a Gal4 tethering system and integrated fluorescent reporter, so the table represents functional protein effector domains rather than genomic enhancer alleles. Coordinates are CDS/transcript-relative and the library is not allele-specific. The table was built from the author’s 195-hit union (EV3), retaining the 131 rows marked as reproducible in both zfh1 replicates and joining replicate call coverage by FlyBase gene/transcript IDs and coordinate overlap. Two retained EV3 regions (dmrt11E and FoxL1) have a reported low cov_pos value in at least one summarized edge call; they were retained because the authors’ final reproducibility flag is the authoritative hit/QC result.

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