PMC

Dose-dependent changes in the distribution of FACT subunits and histone H2B in CBL0137-treated cells. Fluorescent imaging of HT1080 cells expressing either mCherry tagged H2B and GFP-tagged SSRP1 (A) or GFP-tagged H2B and mCherry tagged SPT16. Cells were treated for 1 h and then fixed with 4% paraformaldehyde.

C-trapping of FACT. (A) Immunoblotting of soluble protein extracts and chromatin pellets of HT1080 cells treated with CBL0137 for 1 h, probed with the indicated antibodies. (B) Immunofluorescence staining of HT1080 cells with antibodies to SSRP1. (C) Fluorescent imaging of two live cells expressing GFP-tagged SSRP1 before and after treatment with CBL0137. Top row—HT1080 cell in interphase, 3μM CBL0137, bottom row—DLD1 cell in mitosis, 5 μM CBL0137. (D) Fluorescent imaging of two live HT1080 cells, interphase and mitotic, expressing GFP-tagged SPT16 before and after treatment with CBL0137. Bars are 10μm.

Model of the mechanism of c-trapping. The upper panel shows the scheme of the nucleosome with a standard color code for core histones (H2A-yellow, H2B – red, H3 – blue, H4 – green) and the domain structure of FACT subunits (NTD – N-terminal domain, DD – dimerization domain, MD – middle domain). The lower panels show two phases of c-trapping: i) n(nucleosome)-trapping that occurs via the SPT16 subunit binding to the hexasome of the partially uncoiled nucleosome when one molecule of CBL0137 is bound per ∼10–100 bp of DNA; ii) z(Z-DNA)-trapping that occurs via SSRP1 subunit binding to DNA when the nucleosome is disassembled upon binding of one or more molecules of CBL0137 to every 10 bp of DNA.

Loss of histone from chromatin in CBL0137-treated cells. (A, B) Live cell images of one Hela-H2B-mCherry/GFP-SSRP1 cell in interphase (A) or undergoing mitosis (B) before and after CBL0137 treatment (5 μM). Red arrows indicate loops (possibly DNA) seen in GFP, but not mCherry channels. (C) Immunoblotting of soluble extracts and chromatin pellet of HeLa cells treated with CBL0137, probed with the indicated antibodies. (D) Gel electrophoresis of DNA isolated from nuclei of HeLa cells incubated with CBL0137 followed by digestion with micrococcal nuclease (MN). The actual concentration of CBL0137 in the incubation buffer and its concentration in the corresponding cell culture medium recalculated per nuclei (in the parentheses) are shown.

Binding of SSRP1 to different types of DNA oligonucleotides under cell-free conditions. (A–D) Gel shift assays of (A) SSRP1 or Z-DNA antibody incubated for 20 min at RT with different types of ³²P-labeled double stranded linear oligonucleotides: random—17 bp non-repetitive DNA fragment, indicated repetitive fragments (composition provided in Materials and Methods) or cruciform DNA probe (X); (B) SSRP1 or SPT16 with AmeC probe in the presence or absence of antibodies to SSRP1 (S₁ – 10D1 from Biolegend, S₂ – D-15 and S₃ – D-7 from Santa Cruz Biotechnology); (C) SSRP1 with ³²P-labeled AmeC probe and 10, 30 or 100 times excess of unlabeled AT or AmeC oligonucleotides; (D) CID domain of SSRP1 with ³²P-labeled AT or AmeC probes. Blue arrows – non-specific band, red arrows – SSRP1 shifted band.

Different SSRP1 domains are responsible for c-trapping in CBL0137- and cisplatin-treated cells. (A) Immunoblotting of soluble extracts of HeLa- GFP-SSRP1 cells transduced with control shRNA, SPT16 shRNA or untransduced (mock), treated with CBL0137 for 1 h. (B) Immunoblotting of supernatant and chromatin pellet of reactions consisting of chromatin purified from HeLa cells, recombinant SSRP1 and SPT16 incubated for 20 min in the presence or absence of 30μM CBL0137, which is the equivalent of the cell-based concentration of 3μM at RT. (C) Scheme of domain organization of full length SSRP1 and designations of truncated mutants used in the study. (D) GFP-fluorescence in nuclei of CBL0137- or cisplatin-treated HeLa cells transduced with the indicated constructs. Cells were fixed with 4% paraformaldehyde either 30 min after start of treatment with 3 μM of CBL0137 or 12 h after start of treatment with 200 μg/ml of cisplatin. (E) Immunoblotting of extracts of HT1080 cells transduced with full length SSRP1 and either CID or HMG domain constructs fused with GFP and treated with different concentrations of CBL0137 for 1 h or cisplatin for 8 h. Antibodies to GFP were used for the detection of SSRP1 variants. (F) Immunoblotting of extracts of HT1080 cells expressing full length SSRP1 or ΔCID and transduced with control shRNA or shRNA to SPT16. Cells were treated with different concentrations of CBL0137 for 1 h.

CBL0137 destabilizes nucleosomes and causes FACT binding to chromatin in vitro. (A) Gel electrophoresis of preassembled mononucleosome Nuc207 incubated with different concentrations of CBL0137. (B) Gel electrophoresis of the products of micrococcal nuclease (MN) digestion of polynucleosome, assembled from circular plasmid DNA and recombinant histones, in the presence and absence of 10μM CBL0137. (C and D) Computer modeling of CBL0137 binding to DNA. a and b – interbase pairs distance in the presence (a) and absence (b) of curaxin. (E) Gel electrophoresis of preassembled mononucleosome Nuc207 incubated with different concentrations of FACT (10, 50, 100, 200 nM) and 10 μM CBL0137. (F) In vitro FACT assisted nucleosome assembly is inhibited by CBL0137. Gel electrophoresis of 207 bp DNA fragment incubated with histones and increasing concentration of FACT, with or without CBL0137 (25 μM). The same gels were visualized using UV for ethidium bromide stained (in gel) DNA (left) and fluorescently labeled histones (right). Control panels show reduction of free DNA and increase in bands corresponding to fully assembled nucleosome (Nuc207). Nuc207 control was not incubated with CBL0137. Arrows indicate positions of nucleosome (n), hexasome (h) and tetrasome (t).

CBL0137 treatment causes binding of SSRP1 to genomic region predicted to form Z-DNA and induces conversion of DNA into Z form in cells. (A, B) Genome browser views of SSRP1 binding in control and CBL0137-treated cells detected using ChIP-sequencing approach. Examples of alignment of NGS reads from three independent experiments (1–3) with HT1080 and two (1–2) with MM1.S cells to (A) a region of chromosome 1 showing loss of peaks from gene coding region in HT1080 cells; (B) appearance of a peaks in treated HT1080 and MM1.S cells at a region of chromosomes 7 and 6 with no known genetic features (RefSeq line). Magnified region of chromosome 6 shown below with each nucleotide represented by colored bar, what reveals uniform di-nucleotide sequence under the peak (green box) versus more variable nucleotide composition at the adjacent region. (C, D) Heat plots and histograms of distribution of sequences predicted to form G-quadruplexes (C) or Z-DNA (D) in relation to the center of SSRP1 bound regions in CBL0137-treated cells. Statistical evaluation and correlation with other forms of predicted non-B DNA regions are shown in . (E) Staining of HeLa cells with antibody to Z-DNA. Immunofluorescence imaging. (F) Immunofluorescence staining with Z-DNA antibody (red) of HT1080 cells expressing either nuclear GFP tagged CID or ΔCID treated with CBL0137 (3 μM, 1 h). (G) ImageJ generated fluorescence intensity profiles along yellow lines shown on panel F (from right to left) for GFP and Z-DNA antibody signals in HT1080 cells expression GFP-tagged CID domain of SSRP1 (upper panel) or SSRP1 lacking CID (ΔCID, lower panel).