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Figure 5. Saft functions via the novel and deeply conserved ZAC domain (A) Depiction of CG11247/Saft protein domain organization. Colored bars indicate Saft protein constructs tested in Figures 5D and S6E. (B) Sequence alignment of second ZAC domain helix bundle conserved from arthropod TFs to vertebrate MORC proteins. Protein sequences are labeled with species of origin, brackets indicate gene family. See also Figure S6C and Table S9. (C) AlphaFold structure prediction of conserved second ZAC domain helix bundle. Rainbow coloring indicates orientation from N (blue) to C terminus (red). See also Figure S6B. (D) Dual luciferase assay of DLM3 motif <t>(Table</t> <t>S2),</t> with or without auxin (IAA)-induced depletion of endogenous AID-tagged Saft protein, both conditions with ectopic expression of either <t>Gal4-DNA-binding</t> domain (Gal4-DBD) (transfection control), wild-type Saft, Saft lacking the ZAC domain, or the Saft zinc-finger region only (see Figures 5A and S6D and Table S2). Values of three replicates normalized to enhancer activity only with mock Gal4-DBD transfection. Mean, standard deviation error bars, two-tailed paired Student’s t test. See also Figure S6E.
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Figure 5. Saft functions via the novel and deeply conserved ZAC domain (A) Depiction of CG11247/Saft protein domain organization. Colored bars indicate Saft protein constructs tested in Figures 5D and S6E. (B) Sequence alignment of second ZAC domain helix bundle conserved from arthropod TFs to vertebrate MORC proteins. Protein sequences are labeled with species of origin, brackets indicate gene family. See also Figure S6C and Table S9. (C) AlphaFold structure prediction of conserved second ZAC domain helix bundle. Rainbow coloring indicates orientation from N (blue) to C terminus (red). See also Figure S6B. (D) Dual luciferase assay of DLM3 motif (Table S2), with or without auxin (IAA)-induced depletion of endogenous AID-tagged Saft protein, both conditions with ectopic expression of either Gal4-DNA-binding domain (Gal4-DBD) (transfection control), wild-type Saft, Saft lacking the ZAC domain, or the Saft zinc-finger region only (see Figures 5A and S6D and Table S2). Values of three replicates normalized to enhancer activity only with mock Gal4-DBD transfection. Mean, standard deviation error bars, two-tailed paired Student’s t test. See also Figure S6E.

Journal: Molecular cell

Article Title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression.

doi: 10.1016/j.molcel.2024.10.041

Figure Lengend Snippet: Figure 5. Saft functions via the novel and deeply conserved ZAC domain (A) Depiction of CG11247/Saft protein domain organization. Colored bars indicate Saft protein constructs tested in Figures 5D and S6E. (B) Sequence alignment of second ZAC domain helix bundle conserved from arthropod TFs to vertebrate MORC proteins. Protein sequences are labeled with species of origin, brackets indicate gene family. See also Figure S6C and Table S9. (C) AlphaFold structure prediction of conserved second ZAC domain helix bundle. Rainbow coloring indicates orientation from N (blue) to C terminus (red). See also Figure S6B. (D) Dual luciferase assay of DLM3 motif (Table S2), with or without auxin (IAA)-induced depletion of endogenous AID-tagged Saft protein, both conditions with ectopic expression of either Gal4-DNA-binding domain (Gal4-DBD) (transfection control), wild-type Saft, Saft lacking the ZAC domain, or the Saft zinc-finger region only (see Figures 5A and S6D and Table S2). Values of three replicates normalized to enhancer activity only with mock Gal4-DBD transfection. Mean, standard deviation error bars, two-tailed paired Student’s t test. See also Figure S6E.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pSTARR-seq_fly Arnold et al.41 Addgene #71499 pSTARR-seq_fly-RpS12 Zabidi et al.43 Addgene #71504 pSTARR-seq-DSCP-DLM3 This Study Table S2 pSTARR-seq-RpS12-DLM3 This Study Table S2 Gateway pDONR 221 Vector Invitrogen 12536017 pAGW-dpse-GAL4-DBD Haberle et al.125 Addgene #125153 pBS-Hsp70-Cas9 Addgene Addgene #46294 pU6-BbsI-chiRNA Addgene Addgene #45946 DNA elements individually tested for transcriptional activity This Study Tables S1 and S2 Protein sequences individually tested for transcriptional activity This Study Table S2 Software and Algorithms Benchling N/A https://benchling.com GraphPad version 10.1.1 N/A https://www.graphpad.com R version 3.5.1 N/A https://www.r-project.org/ MEME version 5.1.1 Bailey and Elkan47 https://meme-suite.org/meme/tools/meme FIMO version 5.5.4 Grant et al.126 https://meme-suite.org/meme/tools/fimo Proteome Discoverer version 2.5.0.400 ThermoFisher https://www.thermofisher.com/ MSAmanda version 2.0.0.19924 Dorfer et al.127 https://ms.imp.ac.at/index.php?action=home Tool ptmRS, based on the tool phosphoRS Taus et al.128 https://ms.imp.ac.at/index.php?action=home IMP-apQuant Doblmann et al.129 https://ms.imp.ac.at/index.php?action=home Intensity-based absolute quantification (iBAQ) Schwanh€ausser et al.130 https://ms.imp.ac.at/index.php?action=home PFAM version 36 Mistry et al.75; Paysan-Lafosse et al.76 http://pfam.xfam.org/ HMMER version 3.3.2 Eddy131 http://hmmer.org/ AlphaFold Jumper et al.73; Varadi et al.74 https://alphafold.ebi.ac.uk/ mafft (-linsi, version 7.505) Katoh and Toh132 https://mafft.cbrc.jp/alignment/server/index.html Jalview Waterhouse et al.133 https://www.jalview.org/ NCBI BLAST Altschul et al.134 https://blast.ncbi.nlm.nih.gov/Blast.cgi?

Techniques: Construct, Sequencing, Labeling, Luciferase, Expressing, Binding Assay, Transfection, Control, Activity Assay, Standard Deviation, Two Tailed Test