Journal: bioRxiv

Article Title: Identification and characterization of repressive domains in Drosophila transcription factors

doi: 10.1101/2022.08.26.505062

Figure Lengend Snippet: A . Repressive TFs (R) are modular and can be divided into their DNA-binding domain (DBD) and their repressive domain (RD) which is sufficient to repress a reporter when tethered to it for example via the Gal-UAS system. B . Schematic of the RD-seq pipeline. The candidate library consists of over 200,000 150 bp fragments tiling the coding sequences of 1133 transcription-related genes, which may contain a RD. Candidate fragments were cloned as a Gal4-DBD fusion library. Drosophila S2 cells with an integrated GFP reporter driven by a specific enhancer and core-promoter (CP) pair and with UAS sites upstream of the enhancer were transfected with the candidate library, followed by fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). C. – E . UCSC genome browser tracks for two replicates of RD-seq screens with the zfh1-DSCP reporter cell line. Black bars on the top indicate the entire coding sequence of the respective factor. Shown is the normalized candidate fragment coverage from the fractions of GFP-negative and GFP-positive cells and small black bars indicating the detected RD region. F . Validations of RD-seq hits in comparison to the Gal4-DBD control in the zfh1-DSCP reporter cell line. Shown are the mean fold change (FC) repression values of 3 replicates and standard deviations as error bars. Significance in comparison to the Gal4-DBD control calculated with two-tailed Student T-tests is indicated above bars: * for P≤0.05, ** for P≤ 0.01. G . Comparison between validation FC repression values and average log2 FC in RD-seq for each RD region in the zfh1-DSCP reporter cell line. Pearson correlation coefficient (PCC) is shown.

Article Snippet: The plasmid backbone for the RD-seq candidate library was derived from ptAD-seq-ubi63E-Gal4-DBD ( Arnold et al , 2018 ) by replacing the ubi63E enhancer with the zfh1 enhancer (from pGL3_zfh1_CP-candidate_luc+; Addgene 86391) in between the KpnI (Thermo) and BglII (Thermo) restriction sites (Suppl.

Techniques: Binding Assay, Clone Assay, Transfection, Fluorescence, FACS, Next-Generation Sequencing, Sequencing, Comparison, Control, Two Tailed Test, Biomarker Discovery

Journal: bioRxiv

Article Title: Identification and characterization of repressive domains in Drosophila transcription factors

doi: 10.1101/2022.08.26.505062

Figure Lengend Snippet: A . Frequency histogram of the position of the center of the 50 AA RD within its full-length protein for all 195 RDs. Positions are scaled over the length of the respective protein sequences. B . Pie chart showing the overlap between RDs and intrinsically disordered regions (IDRs) according to the MobiDB-lite database, DNA-binding domains (DBDs), and other annotated protein domains from the Pfam or ProSitePatterns databases. C . Hierarchical clustering of MEME de novo motif discovery motif hits with distinct subsets of RDs (all, global, zfh1, ent1; see methods). The tree was cut at height 0.7, resulting in 11 non-redundant distinct motifs. D . Pie chart showing ELM database and MEME motif instances among the 195 RDs. E . Number of instances of 9 MEME motifs (excluding motif 6 and 10) among the 195 RDs and co-occurrence of these motifs. N indicates the total number of RDs with a certain motif. F. – J . Validation results for wild type and mutated RDs in the zfh1-DSCP reporter cell line with the following conserved motifs: F. Motif 1 – AAxxL, G. Motif 2 – PxDLS, H. Motif 3 – HKKF, I. Motif 4 – PLKKR, J. Motif 5 – EH1. Shown are mean FC repression values of 3 replicates and standard deviations. Significance in comparison to the wild type RDs calculated with two-tailed Student T-tests is indicated above bars: * for P≤0.05, ** for P≤0.01, or exact P-value when not significant.

Article Snippet: The plasmid backbone for the RD-seq candidate library was derived from ptAD-seq-ubi63E-Gal4-DBD ( Arnold et al , 2018 ) by replacing the ubi63E enhancer with the zfh1 enhancer (from pGL3_zfh1_CP-candidate_luc+; Addgene 86391) in between the KpnI (Thermo) and BglII (Thermo) restriction sites (Suppl.

Techniques: Binding Assay, Biomarker Discovery, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Identification and characterization of repressive domains in Drosophila transcription factors

doi: 10.1101/2022.08.26.505062

Figure Lengend Snippet: A . Positioning of RDs and DBDs. Density distribution of the position of the center of the 50 AA RD or the DBD regions within their full-length protein. Positions are scaled over the length of the respective protein sequences. B . Positioning of RDs with distinct motifs from MEME de novo motif searches. Shown are frequency histograms of the position of the center of the 50 AA RD within its full-length protein for all RDs containing each motif type. Positions are scaled over the length of the respective protein sequences. C . Western blots for FLAG-Gal4-DBD-tagged wild type and motif mutant RDs expressed in the zfh1-DSCP reporter cell line. Blots were probed with an anti-Tubulin antibody as loading control.

Article Snippet: The plasmid backbone for the RD-seq candidate library was derived from ptAD-seq-ubi63E-Gal4-DBD ( Arnold et al , 2018 ) by replacing the ubi63E enhancer with the zfh1 enhancer (from pGL3_zfh1_CP-candidate_luc+; Addgene 86391) in between the KpnI (Thermo) and BglII (Thermo) restriction sites (Suppl.

Techniques: Western Blot, Mutagenesis, Control

Journal: bioRxiv

Article Title: Identification and characterization of repressive domains in Drosophila transcription factors

doi: 10.1101/2022.08.26.505062

Figure Lengend Snippet: A. – D . Results of immunoprecipitations followed by mass spectrometry (IP-MS) for pools of RDs with specific repressive motifs: A. PxDLS, B. AAxxL, C. PLKKR, D. HKKF. Shown are volcano plots with the log2FC over control on the x-axis and the -log10 P-value on the y-axis. The FLAG-Gal4-DBD-tagged RDs used as bait for the IPs are indicated in boxes. E. – I . Validations of RDs upon RNAi-mediated depletion of CoRs in the zfh1-DSCP reporter cell line. Each CoR was targeted for depletion with 2 different dsRNA constructs. A dsRNA targeting Renilla and a condition without any dsRNA (noRNA) were used as controls. Shown are means of FC repression values of 3 replicates with standard deviations (E.-G.) or the FC repression value of 1 replicate (H., I.). The repressive motif contained in the tested RD is indicated above the panels. For E.-G. significance in comparison to the noRNA control calculated with two-tailed Student T-tests is indicated above bars: * for P≤0.05, ** for P≤0.01, or not significant (ns) for P>0.05.

Article Snippet: The plasmid backbone for the RD-seq candidate library was derived from ptAD-seq-ubi63E-Gal4-DBD ( Arnold et al , 2018 ) by replacing the ubi63E enhancer with the zfh1 enhancer (from pGL3_zfh1_CP-candidate_luc+; Addgene 86391) in between the KpnI (Thermo) and BglII (Thermo) restriction sites (Suppl.

Techniques: Mass Spectrometry, Protein-Protein interactions, Control, Construct, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Identification and characterization of repressive domains in Drosophila transcription factors

doi: 10.1101/2022.08.26.505062

Figure Lengend Snippet: A . Assessment of depletion of CoR mRNA with RNAi through reverse transcription quantitative PCR (RT-qPCR). Each CoR was targeted with 2 different dsRNA constructs. A dsRNA targeting Renilla was used as a negative control. Shown is the fold change (FC) relative to the control condition for one replicate each calculated with the Delta-Delta Ct Method. B . Repressors with multiple RDs and RDs with multiple repressive motifs. Shown are RD sequences, presence of repressive motifs, their associated interacting CoRs and literature references. C . Validation of wild type and mutant Sna-RD in the zfh1-DSCP reporter cell line. Shown are mean FC repression values of 3 replicates and standard deviations. Significance in comparison to the wild type RD calculated with two-tailed Student T-tests is indicated above bars: * for P≤0.05, ** for P≤0.01, not significant (ns) for P>0.05. D . Validation of Sna-RD upon RNAi-mediated depletion of CtBP in the zfh1-DSCP reporter cell line. CtBP was targeted for depletion with 2 different dsRNA constructs. A dsRNA targeting Renilla and a condition without any dsRNA added (noRNA) were used as controls. Shown are means of FC repression values of 3 replicates with standard deviations. Significance in comparison to the noRNA control calculated with two-tailed Student T-tests is indicated above bars: * for P≤0.05, ** for P≤0.01, or not significant (ns) for P>0.05.

Article Snippet: The plasmid backbone for the RD-seq candidate library was derived from ptAD-seq-ubi63E-Gal4-DBD ( Arnold et al , 2018 ) by replacing the ubi63E enhancer with the zfh1 enhancer (from pGL3_zfh1_CP-candidate_luc+; Addgene 86391) in between the KpnI (Thermo) and BglII (Thermo) restriction sites (Suppl.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Construct, Negative Control, Control, Biomarker Discovery, Mutagenesis, Comparison, Two Tailed Test