Journal: Nature Communications

Article Title: Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA

doi: 10.1038/ncomms2330

Figure Lengend Snippet: Representative images of Alba–DNA complexes visualized by AFM. Nicked pRD24 plasmids are incubated at different stoichiometries (indicated as dimer:bp) with different Alba proteins. ( a – e ) Alba1 forms bridges at 1:60 and 1:30 ratios, condenses molecules at a 1:15 ratio and forms stiff open DNA molecules at 1:7.5 ratio. ( f – j ) Alba1:Alba2 heterodimers form bridged protein–DNA complexes at 1:60, 1:30 and 1:15. However, at 1:7.5 ratio DNA molecules are highly condensed, and do not show a stiffened configuration as Alba1. ( k – o ) Alba1 F60A dimers are able to form bridges between DNA duplexes at all different concentrations. Alba1 F60A and Alba2 both lack the crucial F60 residue and its equivalent. The sequence identity of the α1-helix responsible for dimer–dimer interactions in both proteins is only 36%, which might explain the differences between Alba1 F60A homodimers and Alba1:Alba2 heterodimers. Scale bar, 100 nm.

Article Snippet: The pRD24 plasmid (a pUC19 derivative, containing a Bpu10I recognition site inserted into the multiple cloning site) used for the AFM experiments was propagated in E. coli strain XL10 and purified (Qiagen plasmid midi kit). pRD24 was nicked by digestion with Bpu10l (Fermentas).

Techniques: Incubation, Residue, Sequencing

Journal: Cell reports

Article Title: Perturbations in 3D genome organization can promote acquired drug resistance.

doi: 10.1016/j.celrep.2023.113124

Figure Lengend Snippet: Figure 4. ABCB1 gene activation in RPE-TxR is associated with detachment from the NL (A) Change in NL interactions of ABCB1 and flanking regions in RPE-TxR compared with RPE-TxS, detected by pA-DamID.51 Data are visualized as in Brueckner et al.52 Bottom panels: gene annotation track (hg38); ABCB1 gene is marked in red. Middle panels: pA-DamID tracks of NL interactions in Taxol-sensitive cells (TxS, blue line) and Taxol-resistant cells (TxR, red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Dashlines mark the fifth and 95th percentiles of genome-wide pA-DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis), the pixel shade indicates the rank of the change in pA-DamID score (experimental [TxR] minus control [TxS]) compared with the genome-wide changes in pA-DamID scores across all possible windows of the same size. Only significant changes (within the fifth or the 95th genome-wide quantiles) are colored. Blue: pA-DamID score is highest in control samples; red: pA-DamID score is highest in experimental samples. (Color key genome-wide quantile is shown on the right of the panel.) (B) pA-DamID signals were quantified as described in the method section using genes as units extended of ± 10 kb (n = 2). Every point represents a single gene; ABCB1 is highlighted in red. Graphs represent the pA-DamID Z score signal correlated between Taxol-resistant cells and Taxol-sensitive cells. The two cell lines show a strong overall correlation (Pearson correlation coefficient R = 0.89), but the ABCB1 gene shows a marked difference in pA-DamID signal between the two cell lines. (C) Schematic representation of the ANCHOR3-system53 to visualize the endogenous ABCB1 locus. (D) Z-projection example image of immunofluorescence staining with LaminB1 antibody and the OR3-ABCB1 focus. Scale bar, 2 mm. (E) Quantification of the shortest distance in three-dimensional space from the ANCHOR3-ABCB1 foci to the nuclear lamina in RPE-1 ANCHOR3 TxS and TxR. The analysis is performed as described in STAR Methods. Red bar represents the median of n = 91 TxS and n = 118 TxR cells from three independent ex- periments, ****p < 0.0001, Mann-Whitney test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER HEPES Gibco Cat# 15630080 Prolong Gold Antifade Mountant Invitrogen; Thermo Fisher Scientific Cat# P36934 Lipofectamine RNAiMAX Thermo Fisher Scientific Cat# 13778150 M3814 (DNAPKi) Selleckchem Cat# S8586 Gibson Assembly Master Mix Biolabs Cat# E2611S Taxol (Paclitaxel) Sigma-Aldrich Cat# 33069-62-4 5-Aza-2-deoxycytidine Sigma-Aldrich Cat# A3656 GSK126 Selleckchem Cat# S7061 BIX-01294 Selleckchem Cat# S8006 NEB 5x Taq master mix Biolabs Cat# M0285L Phusion High-Fidelity DNA Polymerase Biolabs Cat# M0530S SSC Buffer 20x Concentrate Sigma-Aldrich Cat# S6639 Formamide (Deionized) Thermo Fisher Scientific Cat# AM9342 Fugene 6 Promega Cat# E5911 DynabeadsTM Protein A Thermo Fisher Scientific Cat# 10001D SuperScriptTM II Reverse Transcriptase Thermo Fisher Scientific Cat# 18064014 Critical commercial assays RNeasy Mini Kit Qiagen Cat# 74104 BioScriptTM Reverse Transcriptase Bioline Cat# BIO-27036 PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 QIAquick Gel Extraction Kit Qiagen Cat# 28704 Dual-Luciferase Reporter Assay System Kit Promega Cat# E1910 ISOLATE II Genomic DNA Kit Meridian Bioscience Cat #BIO-52067 EpiJET Bisulfite Conversion Kit Thermo Fisher Scientific Cat# K1461 MyTaq Red HS Mix Meridian Bioscience Cat# BIO-25046 Experimental models: Cell lines RPE-hTERT iCut van den Berg et al.56 N/A RPE-hTERT CRISPRa Brueckner et al.52 N/A A549 NKI Cryostorage N/A MDA-MB-231 NKI Cryostorage N/A FADU NKI Cryostorage N/A Oligonucleotides sgRNAs for CRISPRa, see Table S1 This paper N/A crRNAs for RPE iCUT KOs, see Table S2 This paper N/A RT-qPCR and ChIP-qPCR primers, see Table S3 This paper N/A RT-qPCR primers position, see Table S4 This paper N/A Recombinant DNA ANCHOR3 plasmids NeoVirTech N/A pUC19 plasmid Addgene Cat# 49793 pSpCas9(BB)-2A-Puro (PX459) Addgene Cat# 62988 pSpCas9(BB)-2A-Puro-ABCB1 This paper N/A pGL3-Basic Vector GenBank Addgene GenBank: U47295 pGL3-Promoter Vector GenBank Addgene GeneBank: U47298 pRL-SV40 Vector GenBank Addgene GeneBank: AF025845 pGL3-ABCB1 This paper N/A (Continued on next page) 18 Cell Reports 42, 113124, October 31, 2023

Techniques: Activation Assay, Genome Wide, Control, Staining, MANN-WHITNEY