Journal: Glia

Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer

doi: 10.1002/glia.23521

Figure Lengend Snippet: (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers.

Article Snippet: Electrophoretic mobility shift assays The DNA binding regions of mouse Sox10 (amino acid 1–235), full-length Olig2, and Ascl1 were cloned into pT7CFE1-cMyc vector (ThermoFisher, USA).

Techniques: Control, Positive Control

Journal:

Article Title: Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System

doi:

Figure Lengend Snippet: Maps of the different DNA segments of the norA promoter examined in this study. The numbers indicate the nucleotide positions according to Yoshida et al. (38). (A) Schematic map of the norA promoter. The −35 and −10 consensus sequences are indicated by black boxes, and the Shine-Dalgarno site is marked SD. The repeated sequences, shown in panel D, are indicated by hatched boxes. (B) PCR fragments used in band shift experiments. (C) Schematic map showing the DNA cloned upstream of the β-lactamase gene in transcriptional fusions. (D) Sequence of the region upstream of the −35 consensus sequence. Repeated sequences are boxed, and the −35 and −10 sequences are underlined.

Article Snippet: To construct a plasmid containing the arlR-arlS locus, a 2.4-kb product containing arlR and arlS was amplified by PCR using Vent DNA polymerase (New England Biolabs), chromosomal DNA of ISP794, and two primers containing the Bam HI site.

Techniques: Electrophoretic Mobility Shift Assay, Clone Assay, Sequencing

Journal:

Article Title: Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System

doi:

Figure Lengend Snippet: Gel mobility shift analysis of the interaction of protein extracts from the wild-type strain ISP794 with different fragments of the norA promoter and the effect of unlabeled DNA. The radiolabeled fragment (arrow) was incubated with increasing amounts of protein extracts. The labeled fragments used in these experiments are L4-R1 (315 bp) (A), L2-R2 (153 bp) (B), L2-R3 (87 bp) (C), L1-R2 (60 bp) (D), and L5-R2 (39 bp) (E). The protein(s) binds to the tested fragment and retards its mobility (a different gel was used for each fragment). An unlabeled fragment of 350 bp amplified by PCR from a Klebsiella oxytoca promoter and an unlabeled fragment of the tested fragment serve as specificity control (NSPE DNA and SPE DNA, respectively). Protein and DNA concentrations and ratios of unlabeled fragments to labeled fragments used in this assay are indicated in the tables above the figures.

Article Snippet: To construct a plasmid containing the arlR-arlS locus, a 2.4-kb product containing arlR and arlS was amplified by PCR using Vent DNA polymerase (New England Biolabs), chromosomal DNA of ISP794, and two primers containing the Bam HI site.

Techniques: Mobility Shift, Incubation, Labeling, Amplification

Journal:

Article Title: Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System

doi:

Figure Lengend Snippet: Isolation of the protein from the wild-type strain ISP794 binding to different fragments of the norA promoter. Different fragments of DNA were immobilized on magnetic beads. Proteins binding to these fragments were then used for different analyses. (A) SDS-PAGE analysis of protein released from DNA affinity magnetic beads. Lane 1, standard proteins (in kilodaltons); lane 2, fragment L2-R2; lane 3, fragment L1-R2; lane 4, fragment L2-R3. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified extracts from strain ISP794. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of ISP794. Free DNA is indicated by an arrow.

Article Snippet: To construct a plasmid containing the arlR-arlS locus, a 2.4-kb product containing arlR and arlS was amplified by PCR using Vent DNA polymerase (New England Biolabs), chromosomal DNA of ISP794, and two primers containing the Bam HI site.

Techniques: Isolation, Binding Assay, Magnetic Beads, SDS Page, Mobility Shift, Affinity Purification, Purification

Journal:

Article Title: Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System

doi:

Figure Lengend Snippet: Isolation of the protein from the mutant BF15 binding to the fragment L2-R2. (A) SDS-PAGE analysis of protein released from affinity-purified extracts from different strains. Lane 1, standard proteins (in kilodaltons); lane 2, purified protein from ISP794; lane 3, purified protein from BF15. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified protein extracts from BF15 and fragment L2-R2. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of BF15. Free DNA is indicated by an arrow.

Article Snippet: To construct a plasmid containing the arlR-arlS locus, a 2.4-kb product containing arlR and arlS was amplified by PCR using Vent DNA polymerase (New England Biolabs), chromosomal DNA of ISP794, and two primers containing the Bam HI site.

Techniques: Isolation, Mutagenesis, Binding Assay, SDS Page, Affinity Purification, Purification, Mobility Shift

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

doi: 10.1161/JAHA.121.023517

Figure Lengend Snippet: A , A representative standard 12‐lead ECG from the proband affected with atrial fibrillation. B , Sequencing chromatograms of the affected proband and an unaffected control subject, exhibiting the heterozygous PRRX1 mutation and its wild‐type control. An arrow symbol points to the heterozygous nucleotides of C/T in the affected proband (mutant type) or the homozygous nucleotides of C/C in an unaffected subject (wild type). Sequencing analysis unveiled a heterozygous mutation of c.319C>T in PRRX1 , resulting in a substitution of a premature stop codon for the glutamine (Gln)–encoding codon at amino acid 107 of PRRX1 (PRRX1‐Gln107*).

Article Snippet: The yielded PRRX1 cDNA was subjected to double digestion with restriction endonucleases Eco RI and Xho I (NEB, Ipswich, MA), and then inserted into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) at the Eco RI‐ Xho I sites to construct the eukaryotic gene expression plasmid PRRX1‐pcDNA3.1.

Techniques: Sequencing, Control, Mutagenesis

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

doi: 10.1161/JAHA.121.023517

Figure Lengend Snippet: A , A representative standard 12‐lead ECG from the index patient experiencing atrial fibrillation. B , Sequencing electropherograms of the index patient and a healthy control individual, with the identified PRRX1‐Arg146Ile mutation indicated by an arrow. Sequence analysis revealed a heterozygous mutation of c.437G>T in PRRX1 , leading to a transversion of arginine (Arg) into isoleucine (Ile) at amino acid 146 of PRRX1 (PRRX1‐Arg146Ile).

Article Snippet: The yielded PRRX1 cDNA was subjected to double digestion with restriction endonucleases Eco RI and Xho I (NEB, Ipswich, MA), and then inserted into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) at the Eco RI‐ Xho I sites to construct the eukaryotic gene expression plasmid PRRX1‐pcDNA3.1.

Techniques: Sequencing, Control, Mutagenesis

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

doi: 10.1161/JAHA.121.023517

Figure Lengend Snippet: A , In cultured HeLa cells, PRRX1 transcriptionally activated the SHOX2 promoter in a dose‐dependent manner. B , Transactivation of the SHOX2 promoter‐driven luciferase by wild‐type PRRX1 (WT) or Gln107*‐mutant PRRX1 (Q107*) or R146I‐mutant PRRX1 (R146I), alone or in combination, showed that Gln107* or R146I had a significantly reduced transcriptional activity. C , In cultured HeLa cells, PRRX1 transcriptionally activated the ISL1 promoter in a dose‐dependent mode. D , Transactivation of the ISL1 promoter‐driven luciferase by WT or Q107* or R146I, alone or in combination, showed that Gln107* or R146I had a significantly decreased transcriptional activity. Transfection experiments were performed in triplicate, and the results are expressed as means±SD. All the letters from a to h indicate P <0.001, when compared with the same amounts of WT.

Article Snippet: The yielded PRRX1 cDNA was subjected to double digestion with restriction endonucleases Eco RI and Xho I (NEB, Ipswich, MA), and then inserted into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) at the Eco RI‐ Xho I sites to construct the eukaryotic gene expression plasmid PRRX1‐pcDNA3.1.

Techniques: Cell Culture, Luciferase, Mutagenesis, Activity Assay, Transfection

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

doi: 10.1161/JAHA.121.023517

Figure Lengend Snippet: A , The ability of wild‐type PRRX1 (WT), Gln107*‐mutant PRRX1 (Q107*), or R146I‐mutant PRRX1 (R146I) to bind the ISL1 promoter DNA. B , The ability of WT, Q107*, or R146I to bind the SHOX2 promoter DNA. Electrophoretic mobility shift assay showed that WT normally bound the ISL1 promoter DNA ( A ) or the SHOX2 promoter DNA ( B ), but either Q107* or R146I had a significantly decreased DNA‐binding affinity for the ISL1 promoter DNA ( A ) or the SHOX2 promoter DNA ( B ), when compared with WT.

Article Snippet: The yielded PRRX1 cDNA was subjected to double digestion with restriction endonucleases Eco RI and Xho I (NEB, Ipswich, MA), and then inserted into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) at the Eco RI‐ Xho I sites to construct the eukaryotic gene expression plasmid PRRX1‐pcDNA3.1.

Techniques: Mutagenesis, Electrophoretic Mobility Shift Assay, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

doi: 10.1161/JAHA.121.023517

Figure Lengend Snippet: Subcellular localizations of wild‐type PRRX1 (WT), Gln107*‐mutant PRRX1 (Q107*), and R146I‐mutant PRRX1 (R146I) were determined by immunofluorescence in HeLa cells. HeLa cells, transfected with WT, Q107*, or R146I, were stained with anti‑FLAG (red) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Enlarged images of FLAG and DAPI staining and merged images of HeLa cells were presented. WT, Q107*, or R146I was localized exclusively to the nuclei with normal subcellular distribution. Bar=100 μm.

Article Snippet: The yielded PRRX1 cDNA was subjected to double digestion with restriction endonucleases Eco RI and Xho I (NEB, Ipswich, MA), and then inserted into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) at the Eco RI‐ Xho I sites to construct the eukaryotic gene expression plasmid PRRX1‐pcDNA3.1.

Techniques: Mutagenesis, Immunofluorescence, Transfection, Staining

Journal: Oncology Letters

Article Title: Piceatannol inhibits MMP-9-dependent invasion of tumor necrosis factor-?-stimulated DU145 cells by suppressing the Akt-mediated nuclear factor-?B pathway

doi: 10.3892/ol.2012.968

Figure Lengend Snippet: Effects of PIC on the viability of DU145 cells. DU145 cells treated with the indicated concentrations of PIC and/or 20 ng/ml TNF-α in the presence (upper panel) or absence of serum (lower panel) for 24 h. (A) Cell viability was measured by an MTT assay following 24 h. (B) DNA content was analyzed by flow cytometry. Percentages of sub-G1 DNA content are represented in each panel. The data and arrow in the figure represent the percentage of each sub-G1 phase in cell cycle distribution. (C) Total DNA was extracted from the treated cells and the DNA fragmentation assay analyzed on a 1.5% agarose gel. (D) For the wound healing assay, cells were treated with PIC and/or PIC/TNF-α in a 6-well plate and incubated for 24 h. Images of the wound were captured under ×100 magnifications using a microscope. Data from 3 independent experiments are expressed as overall mean ± SE. Statistical significance was determined by one-way ANOVA test (*P<0.05 vs. untreated control). PIC, piceatannol; TNF-α, tumor necrosis factor-α; UT, untreated.

Article Snippet: Human prostate cancer DU145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: MTT Assay, Flow Cytometry, DNA Fragmentation Assay, Agarose Gel Electrophoresis, Wound Healing Assay, Incubation, Microscopy, Control

Journal: Oncology Letters

Article Title: Piceatannol inhibits MMP-9-dependent invasion of tumor necrosis factor-?-stimulated DU145 cells by suppressing the Akt-mediated nuclear factor-?B pathway

doi: 10.3892/ol.2012.968

Figure Lengend Snippet: Effects of PIC on MMP-9 activity and invasion. DU145 cells were treated with various concentrations of PIC in the presence of TNF-α (20 ng/ml). (A) Conditioned medium was collected from cultures following 24 h and analyzed by gelatin zymography. (B) In a parallel experiment, DU145 cells were transfected with a WT-MMP-9 promoter-containing reporter vector and luciferase activity was measured 24 h following transfection. (C) Total RNA was isolated and RT-PCR analysis was performed. (D) For the invasion assay, the lower and upper parts of the Transwells were coated with Matrigel. DU145 cells were cultured with PIC or MMP-9 inhibitor I (MMP-9-I; 5 nM) in the presence or absence of TNF-α. Invasiveness of the cells was determined by measuring their ability to pass through a layer of a Matrigel-coated filter. Following 24 h, cells on the bottom side of the filter were fixed, stained and counted as described in Materials and methods. Data from 3 independent experiments are expressed as overall mean ± SE. Statistical significance was determined by one-way ANOVA test (*P<0.05 vs. TNF-α-treated group). PIC, piceatannol; TNF-α, tumor necrosis factor-α; MMP, matrix metalloproteinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human prostate cancer DU145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Zymography, Transfection, Plasmid Preparation, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Cell Culture, Staining

Journal: Oncology Letters

Article Title: Piceatannol inhibits MMP-9-dependent invasion of tumor necrosis factor-?-stimulated DU145 cells by suppressing the Akt-mediated nuclear factor-?B pathway

doi: 10.3892/ol.2012.968

Figure Lengend Snippet: Effects of PIC on NF-κB-dependent MMP-9 regulation. DU145 cells were treated with 40 μM PIC in the presence of TNF-α (20 ng/ml). (A) Cells were transfected with an NF-κB promoter-containing reporter vector and luciferase activity was measured 24 h following transfection. (B) The nuclear extracts were prepared at 30 min and the EMSA measured NF-κB binding to its DNA promoter region in the extract. (C) The cytosolic extracts were prepared to determine the levels of p65, p50 and IκBα by western blot analysis at 30 min. (D) Cells (1×105 cells/ml) were incubated with 30 μM PDTC 1 h before TNF-α (20 ng/ml) treatment for 18 h. Total RNA was isolated and RT-PCR analysis of MMP-9 was performed. GAPDH was used as an internal control for RT-PCR. Data from 3 independent experiments are expressed as overall mean ± SE. Statistical significance was determined by one-way ANOVA test (*P<0.05 vs. TNF-α-treated group). PIC, piceatannol; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidine dithiocarbamate; MMP, matrix metalloproteinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human prostate cancer DU145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Western Blot, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Electrophoretic Mobility Shift Assay

Journal: Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society

Article Title: Rapamycin inhibits cholangiocyte regeneration by blocking interleukin-6-induced activation of signal transducer and activator of transcription 3 after liver transplantation.

doi: 10.1002/lt.21985

Figure Lengend Snippet: Figure 4. Western blotting for p-STAT3 and cyclin D1 expression in cholangiocytes. (A) Each lane shows a representative extract and change for p-STAT3 and cyclin D1. (B,C) Data are expressed with respect to the b-actin level as means and standard deviations. The OLT-12h group showed much higher expression of p-STAT3 and cyclin D1 than the other groups up to 7 days after the operation. In the anti–sIL-6R and RPM groups, p-STAT3 and cyclin D1 expression was significantly lower than that in the OLT-12h group until the end of observation. aP < 0.05 versus the sham group, bP < 0.05 versus the OLT-1h group, and cP < 0.05 versus the OLT-12h group.

Article Snippet: A rat IL-6 enzyme-linked immunosorbent assay kit was bought from Shenzhen Jingmei Biotech Co. A goat anti–Ki-67 monoclonal antibody (sc-7846), a goat anti–p-STAT3 (Ser727) monoclonal antibody (sc21876), a mouse anti–cyclin D1 antibody (sc-70899), and a goat anti–cytokeratin 19 antibody (sc-33119) were bought from Santa Cruz (United States).

Techniques: Western Blot, Expressing

Journal: Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society

Article Title: Rapamycin inhibits cholangiocyte regeneration by blocking interleukin-6-induced activation of signal transducer and activator of transcription 3 after liver transplantation.

doi: 10.1002/lt.21985

Figure Lengend Snippet: Figure 5. STAT3 DNA binding assay by electrophoretic mobility shift assays. Each lane shows representative isolated cholangiocytes from an individual animal from one of the groups sacrificed at a special time point following surgery. The DNA binding activity of STAT3 was much higher in the OLT-12h group than the activity in the other groups, with the peak at 1 day after transplantation. Anti– sIL-6R and RPM potently inhibited STAT3 activation in the grafts with 12 hours of cold preservation.

Article Snippet: A rat IL-6 enzyme-linked immunosorbent assay kit was bought from Shenzhen Jingmei Biotech Co. A goat anti–Ki-67 monoclonal antibody (sc-7846), a goat anti–p-STAT3 (Ser727) monoclonal antibody (sc21876), a mouse anti–cyclin D1 antibody (sc-70899), and a goat anti–cytokeratin 19 antibody (sc-33119) were bought from Santa Cruz (United States).

Techniques: DNA Binding Assay, Electrophoretic Mobility Shift Assay, Isolation, Binding Assay, Activity Assay, Transplantation Assay, Activation Assay, Preserving

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: Immunohistochemistry images of testis samples are shown from male mouse foetuses. a , Z-DNA antibody staining (green) is diminishing in germ cells, as identified by PGC7 staining (red) at 13.5, 15.5 and 18.5 dpc. Scale bars, 5 µm. b , c , Loss of Z-DNA is specific to the germ cells in the foetal testis. Immunohistochemistry is shown for foetal testis sections at 13.5, 15.5 and 18.5 dpc timepoints, using Z-DNA antibody (green), germ cell marker (PGC7) and DAPI counterstain. Merged images are displayed on the right. Prospermatogonia ( b ) exhibit loss of Z-DNA. Somatic cells ( c ) maintain Z-DNA. Scale bars, 1 µm. d , ZBTB43 protein is detected in germ cells inside the testicular cords of the wild-type but not in the Zbtb43 −/− mutant testis at 15.5 dpc. Note the typical weak and diffuse DAPI staining of germ cell nuclei. The surrounding Sertoli cells, which exhibit stronger DAPI staining, are negative for ZBTB43 staining. Scale bars, 5 µm. e , Double staining of 15.5 dpc germ cells with ZBTB43 and germ cell marker OCT4 antibodies is shown in testis samples at 15.5 dpc. Scale bars, 2 µm. f , Double staining of testis samples using the ZBTB43 and germ cell marker DDX4 antibodies is shown at the foetal days as marked. Scale bars, 2 µm. The results shown represent three independent immunostaining experiments using four biologically independent testis samples per experiment ( a – c ), five independent experiments using three biologically independent testis samples per experiment ( d ), one immunostaining experiment using two biologically independent testis samples ( e ) and two independent immunostaining experiments using two biologically independent testis samples per experiment ( f ).

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Immunohistochemistry, Staining, Marker, Mutagenesis, Double Staining, Immunostaining

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) The fetal testis and mesonephros are shown in transmitted light to the left. Testicular cords are visualized by transgenic EGFP expression to the right. EGFP expression in the TgOG2 mouse line allows germ cell isolation by FACS. MGC: male germ cell (EGFP positive); MSC: male somatic cell (EGFP negative). FACS gating strategy is shown. R6: EGFP+, R5: EGFP-. ( b ) RNA-seq results are displayed in purified male germ cells (MGC), female germ cells (FGC) and somatic cells of male and female gonads (MSC and FSC) at 15.5 dpc ( GSE46953 ). Transcript levels of Zbtb43 are plotted among other Zbtb family members. ( c ) Zbtb43 expression is depicted among some of the most highly expressed transcripts (apart from ribosomal proteins, elongation complex members or chaperons) in purified MGC, FGC, MSC, and FSC at 15.5 dpc. ( d ) Zbtb43 expression is shown for mouse organs outside of the gonads. GEO Profiles: GDS661/116970_at from #BioProject PRJNA66167.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Transgenic Assay, Expressing, Cell Isolation, RNA Sequencing Assay, Purification

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Knockout strategy. Mice carrying the Zbtb43 tm1b(KOMP)Mbp allele. A LacZ cassette replaced exon 4 of the Zbtb43 gene. ( b ) PCR genotyping from tail DNA to distinguish Zbtb43 +/+ , Zbtb43 +/- and Zbtb43 -/- pups at weaning. Data shown represent three independent experiments. ( c ) Western blot in adult kidney samples using the anti-ZBTB43 antibody and GAPDH loading control. ( d ) Western blot in adult kidney and brain samples using the anti-LacZ antibody. Data shown represent two independent experiments in c and d.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Knock-Out, Western Blot

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Structure of the ZBTB43 protein. Location of the BTB domain and the three ZF domains are indicated. The full-length protein (ZBTB43-FL), its ZF domain (ZBTB43-ZF) and its BTB domain (ZBTB43-BTB) were purified, as shown in the protein gels to the right. Protein purification and protein gel testing was done twice for ZBTB-FL and ZBTB-ZF and once for ZBTB43-BTB. b , Outline of the affinity sequencing experiment. MBP tag was used for the capture. c , ZBTB43 protein has affinity to methylated and unmethylated genomic DNA. Heatmap analysis of MBP-ZBTB43-FL binding is shown to genomic DNA, either DNMT-TKO ES cell DNA or DNA fully methylated by SssI bacterial CpG methyltransferase centred at the TKO DNA peaks. Background level binding by MBP is shown on the right. d , Consensus binding sequences of the affinity peaks in TKO determined by RSAT (top), significance 4.5e −13 , BaMM (middle), dataset performance 0.096, motif performance 0.84, and MEME (bottom), significance 4.7e −2775 . e , Heatmap shows the match between ZBTB43 affinity binding and predicted Z-DNA sites . f , IGV browser images of selected specific MBP-ZBTB43 peaks are shown in TKO DNA samples at four genomic regions. Control samples show the background of MBP capture. The tracks for transcripts and predicted Z-DNA are displayed at the bottom. The affinity-sequencing results shown represent two independent biological replicates in c , e and f . g , EMSA confirm the binding of ZBTB43-FL or ZBTB43-ZF to the regions detected by affinity sequencing. The FAM-labelled probes (CACG) 8 , Rps6kl1 , and Ago2 , marked as a, b, and c, respectively, were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. ( h) EMSA confirms the binding of ZBTB43-ZF to the consensus PPR sequences. The FAM-labelled probes (CA) and (CACG) 8 , marked as d and a, respectively, resulted in specific shift; they were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. The BTB domain of ZBTB43 (ZBTB43-BTB) lacked binding activity. Data shown represent three independent experiments in panels g and h.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Purification, Protein Purification, Sequencing, Capture-C, Methylation, Binding Assay, Mutagenesis, Activity Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a-b ) Heatmap analysis is shown of MBP-ZBTB43 (FL) binding intensities to genomic DNA, either Dnmt1/Dnmt3a/Dnmt3b triple knockout (TKO) ES cell DNA or DNA fully methylated by SssI bacterial CpG methyltransferase (SssI) centered at the TKO DNA peaks ( a ) and at the SssI peaks ( b ). Background level binding by MBP is included to the right. ( c ) Venn diagram shows the relationship between ZBTB43 affinity peaks mapped in SssI and TKO DNA. ( d ) Venn diagram shows the relationship between ZBTB43 affinity peaks mapped in SssI DNA, TKO DNA, and predicted Z-DNA sites or enriched Z-DNA sites in activated B-cells . ( e-f ) Heatmaps show the match between ZBTB43 affinity binding peaks and Z-DNA sites. The affinity intensities are centered at predicted Z-DNA sites ( e ) and enriched Z-DNA sites mapped in activated B-cells ( f ). ( g-h ) Venn diagrams displays the relationship between predicted Z-DNA locations and ZBTB43 affinity binding sites in SssI ( g ) or TKO DNA ( h ). ( i-j ) Venn diagrams displays the relationship between in vivo mapped Z-DNA sites in activated B-cells and ZBTB43 affinity binding sites in SssI DNA ( i ) or TKO DNA ( j ). Affinity-sequencing results shown represent two biologically independent samples.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Binding Assay, Triple Knockout, Methylation, In Vivo, Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: Venn diagrams depict the location of ZBTB43 affinity peaks mapped in fully methylated genomic DNA (SssI) and unmethylated genomic DNA (TKO) relative to the location of transcripts in the genome. In comparison, the distribution of predicted Z-DNA and enriched Z-DNA mapped in vivo in activated B-cells are also depicted to the right. ( a ) All genomic locations are plotted. ( b ) Distal intergenic regions are excluded. Affinity-sequencing results represent two independent biological replicates.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Methylation, In Vivo, Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: IGV browser images of selected specific MBP-ZBTB43 peaks are shown in unmethylated genomic DNA (TKO) at four genomic regions. Control samples show the background of MBP capture. Independent biological replicate samples are displayed. The tracks for transcripts and predicted Z-DNA are displayed at the bottom. The predicted Z-DNA sequence is enlarged to illustrate the underlying PPR sequences. The ZBTB43 peaks are often found in an intron of very long transcripts. ( a ) Rps6kl1 ( b ) Ago2 ( c ) Arid2 ( d ) Eml1 .

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , ZBTB43 binding sequence at Rps6kl1 , in forward and reverse orientation (Z1 and Z2), was inserted into the vector pUCON, and the resulting plasmids pUPZ1 and pUPZ2 were tested in LacZ mutation assays. b , Mutation frequencies in bacterial NEB Stable cells. Average values of three replicate samples are shown with standard deviation. Statistically significant differences ( P < 0.05) resulting from n = 3 independent experiments are marked. These were obtained using two-tailed Student’s t -tests (unequal variance). c , The LacZ gene is frequently mutated at the Z-DNA insert, as depicted by an agarose plate with DH5α bacteria that contain the parent plasmid (blue colonies) or its mutants (white colonies). d , Restriction digestion is shown from randomly picked clones recovered from DH5α bacteria. DNA sequencing revealed small mutations that did not change the size of the 910-bp-long PPR-containing DNA fragment (blue arrow). Molecular size marker is shown in the left lane. Wild-type plasmid (WT) is depicted in the right lane. e , Mutation frequencies in mammalian COS-7 cells. Average values of three replicate samples are shown with standard deviation. Statistically significant differences ( P < 0.05) resulting from n = 3 independent experiments are marked. These were obtained using two-tailed Student’s t -tests (unequal variance). f , Restriction digestion is shown from randomly picked biologically independent clones recovered from COS-7 cells and grown in NEB Stable or DH5α bacteria. The loss of the 910-bp-long PPR-containing fragment (blue arrow) reveals large deletions. Molecular size marker is shown in the left lane. Wild-type plasmid (WT) is depicted in the right lane. Restriction digestion results in d and f are shown from one of three independent mutagenesis experiments. g , Mutation detection in the pUPZ1 and pUPZ2 plasmids mutagenized in COS-7 cells. Structural elements of the vector plasmid are depicted at the top. Position of the inserted Z1 and reciprocal, Z2 sequences is shown in turquoise. Sequencing results are shown from biologically independent clones recovered in NEB Stable or DH5α bacteria, as indicated. Deletions are marked by blue horizontal bars. Micro-homologies that flank these deletions are shown by the DNA sequence. Sequencing results are shown from one of three independent mutagenesis experiments.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Binding Assay, Sequencing, Plasmid Preparation, Mutagenesis, Standard Deviation, Two Tailed Test, Clone Assay, DNA Sequencing, Marker

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , EMSA. Hexamine CoCl 3 was added to the DNA probes at increasing concentrations. The induced Z-DNA was detected as a gel shift using the anti-Z-DNA antibody Z22. b , Z-DNA formation (blue lines) is detected by CD spectroscopy at the (CACG) 8 consensus sequence in response to different concentrations of CoCl 3 . The B-DNA specific spectrum is displayed in red. c , 2D gel electrophoresis detects a kink (blue arrow) at certain plasmid topoisomers, a sign of Z-DNA formation. d , Generation of circular Z-DNA probe. When two single-stranded circles (CC) are annealed, part of the circle, which contains PPRs is forced into left-handed DNA . Circular B-DNA is prepared by annealing a circular and a linear strand followed by ligating the nick (CL). Linear B-DNA is prepared by annealing two strands of linear DNA (LL) . e , f , Z-DNA structure is confirmed in the CC probe by its insensitivity to restriction enzymes. The CC, CL and LL probes of PPR sequences, as marked above, were subjected to restriction digestion. The CC form (turquoise asterisk), was refractory. The CL form (black asterisk) is linearized, and the LL form is restricted to two fragments (red asterisks). Experiments where the (CACG) 6 sequence ( e ), and the Rps6kl1 -Y peak sequence ( f ) are digested using Hha I and Bsiw I are displayed. g , Z-DNA is formed in the CC probe at the ZBTB43 consensus sequence. Increasing amount of the Z-DNA antibody Z22 quantitatively shifts the CC probe of (CACG) 6 and Rps6kl1 -Z sequences. h , ZBTB43 binds Z-DNA. EMSA results show that ZBTB43-FL shifts the CC probe containing the (CACG) 6 and Rps6kl1 -Z sequences. Data shown represent three independent experiments in a – c and e – h . i , ZBTB43 binds both Z-DNA and B-DNA but prefers Z-DNA. Competition binding experiment is shown where the CC and CL probes were mixed at equal molar ratios and the aliquots were allowed to interact with increasing amounts of ZBTB43-FL before separating the free probes and complexes in EMSA gels. The remaining free CC and CL probes in each reaction were quantified in the gel images. Data are presented as mean ± standard error of the mean (s.e.m.) from n = 3 independent experiments.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Electrophoretic Mobility Shift Assay, Spectroscopy, Sequencing, Two-Dimensional Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Binding Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a – c show that ZBTB43 has the capacity to remove Z-DNA in vitro. a , Restriction digestion using Tai I of the Rps6kl1 -Z affinity peak in the CC probe (blue asterisk) and the LL probe confirms Z-DNA and B-DNA, respectively. b , ZBTB43 renders CC sensitive to digestion. The CC probe was reacted with TOPO1, or ZBTB43, or both, followed by phenol extraction and precipitation. The resulting DNA was run on a gel before or after Tai I digestion. TOPO1 and/or ZBTB43 resulted in a band that migrated slower than the CC form (black asterisk). Tai I produced a linear 121 bp fragment (red asterisk). c , ZBTB43 has no effect on B-DNA. The LL probe (121 bp) was reacted with TOPO1, and/or ZBTB43, and the recovered LL DNA was still sensitive to Tai I digestion into two fragments of 37 and 84 bp. d , ZBTB43 enhances the effect of TOPO1 in reducing supercoiling-induced tension. TOPO1 was reacted with plasmid DNA containing the Rps6kl1 affinity peak sequence in the presence or absence of ethidium bromide (EB6 or EB0, respectively) and increasing amounts of ZBTB43-FL. Control reactions were run without TOPO1. Supercoiled plasmid (PL) purified from bacteria and a molecular weight marker (M) are included. e – h show that ZBTB43 reverses the action of ADAR1 Z-α domain on DNA topology in vitro. e , To induce Z-DNA, the Z-α domain was added to the (CA) 16 linear DNA probe in increasing molar excess. B-DNA specific spectrum, peaking at 280 nm (orange dot) is eliminated by 20× excess of Z-α. New Z-DNA peaks (turquoise and blue dots) at 260 nm are visible at 40× and 80× excess of Z-α. f , ZBTB43 has no effect on B-DNA topology. g , Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 20-fold molar excess of Z-α (280 nm peak regained, green dot). h , Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 40-fold molar excess of Z-α (ZBTB43 reverts the peak from 260 nm). Data shown represent two ( a – c ) or three ( d ) independent experiments. Experiments in e – h have been performed once, and each CD spectrum is presented as an average of three scans.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: In Vitro, Produced, Plasmid Preparation, Sequencing, Purification, Molecular Weight, Marker

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Global Z-DNA is eliminated by ZBTB43 in Zbtb43 +/+ , but not in Zbtb43 −/− foetal testis sections at 15.5 dpc. Images of testicular cords were obtained by immunohistochemistry and confocal microscopy using the ZBTB43 (red), and Z-DNA (green) antibodies, counterstained with DAPI. Scale bars, 10 µm. b , Images of testicular cords as above are shown using higher magnification and background reduction. Scale bar, 5 µm. c , Images of germ cells as above are shown using higher magnification. Scale bars, 2 µm. d , Quantification of the fluorescence intensities of DAPI, Z-DNA and ZBTB43 immunostaining are depicted in 15.5 dpc Zbtb43 +/+ and Zbtb43 −/− prospermatogonia. Spermatogonia ( n = 10 + 9) were quantified from two independent foetuses for each genotype. The intensity was measured by Fiji, and the quantification was done by Prism. Data are presented as mean ± s.e.m. The differences between genotypes were statistically significant for the Z-DNA and ZBTB43 intensities by multiple unpaired two-tailed t -tests. e , ZBTB43 protects from DSBs. Immunohistochemistry of the 15.5 dpc Zbtb43 +/+ and Zbtb43 −/− foetal testis sections is shown using the γH2AX (red) and DDX4 (green) antibodies, counterstained with DAPI. Scale bars, 5 µm. f , Enlarged details of the testicular cords are displayed. Scale bars, 2 µm. The results shown represent three ( a – c ) or two ( e , f ) independent experiments done using four biologically independent testis samples per experiment.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Immunohistochemistry, Confocal Microscopy, Fluorescence, Immunostaining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a-b ) Immunohistochemistry is shown of the Zbtb43 +/+ wild-type ( a ), and Zbtb43 -/- mutant ( b ) fetal testis sections at 13.5, 15.5 and 18.5 dpc using the ZBTB43 (red), and Z-DNA (green) antibodies, counterstained with DAPI. Scale bar: 10 µm. The ZBTB43-Z-DNA double staining was done three times at all stages. ( c-e ) Quantification of signal intensities of DAPI, Z-DNA and ZBTB43 in Zbtb43 +/+ wild-type, and Zbtb43 -/- mutant fetal male germ cells shown relative to the corresponding somatic cells at 13.5 dpc (n=10 cells from one out of two independent experiments) and at 15.5 and 18.5 dpc (n=19 cells from one out of two independent experiments). Data are presented as mean values +/- SEM. Statistically significant changes as calculated by multiple 2-sided T-tests are marked with P -value.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Immunohistochemistry, Mutagenesis, Double Staining

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Genome browser images of ZBTB43 ChIP–seq peaks (light green) obtained in purified 15.5 dpc prospermatogonia are displayed at 12 genomic locations. IgG lanes are shown as controls. The ChIP peaks align with the in vitro affinity sequencing peaks found in fully methylated genomic DNA (SssI) and in fully unmethylated genomic DNA (TKO) (dark green). MBP affinity-seq lanes are shown as controls. The scale of reads was normalized between experimental samples and their respective background control samples using the ‘group-autoscale’ function of IGV as marked on the right. b , c , Heatmap showing the ZBTB43 ChIP–seq peaks detected against IgG background in 100,000 ( b ) or 500,000 ( c ) purified prospermatogonia. The read intensities in three libraries, ZBTB43 antibody, IgG antibody and input DNA (as marked above), are plotted centred at the peak and using +1 kb and −1 kb flanking regions. d , e , Venn diagrams depicting the location of ZBTB43 ChIP–seq peaks mapped in 15.5 dpc prospermatogonia relative to the location of transcripts in the genome. In d , all genomic locations are plotted. In e , distal intergenic regions are excluded. f , The ZBTB43 ChIP–seq peaks detected in vivo are recognized by purified ZBTB43 protein in vitro. Heatmaps display the read intensities of ZBTB43-FL and the control MBP in affinity binding with unmethylated DNA (TKO) or methylated DNA (SssI). The plotted regions were centred at ChIP peaks called in 500,000 or 100,000 prospermatogonia against IgG, as indicated at the bottom. g , The ZBTB43 ChIP–seq peaks detected in prospermatogonia align with affinity-seq peaks and with predicted Z-DNA. Heat maps display the ChIP–seq log 2 IP/IgG read intensities detected in 500 K or 100 K prospermatogonia (as marked at the top) along subset of genomic regions centred at ZBTB43-FL affinity-seq peaks in methylated DNA (SssI), unmethylated DNA (TKO) and at the subset of predicted Z-DNA sites (marked at the bottom) where overlap is found with ChIP–seq peaks. The ChIP–seq and affinity-sequencing results shown represent two independent biological replicates in a , f and g .

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: ChIP-sequencing, Purification, In Vitro, Sequencing, Methylation, In Vivo, Binding Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Z-DNA structure inhibits DNMT3A catalytic activity in vitro. The CC, CL and LL substrates were prepared from the Rps6kl1 affinity binding sequence and were subjected to methylation by DNMT3A at 30× or 60× molar excess. The level of DNA methylation at each CpG is plotted after multiplexed bisulfite sequencing in two biologically independent replicates. b , DNA methylation is aberrant in sperm of Zbtb43 −/− mice at the sites of ZBTB43 binding. IGV browser images display selected in vivo hypomethylated regions detected using MIRA–seq assays in Zbtb43 −/− versus wild-type spermatozoa (navy). The ZBTB43 affinity binding peaks (green), and ChIP–seq peaks mapped in 15.5 dpc prospermatogonia (light green) are also shown together with the MBP and IgG backgrounds. Independent replicate samples are displayed. c , DMRs originate at the time of de novo methylation in prospermatogonia. Bisulfite sequencing results show methylated and unmethylated CpGs (black and white circle, respectively) along individual chromosomes at specific DMR sequences and at one control region in adult spermatozoa (top) and 18.5 dpc prospermatogonia (bottom). The genotypes are marked on the left. d , Heatmap displaying the MIRA–seq intensities in navy at the hypomethylated and hypermethylated DMRs identified between Zbtb43 −/− and Zbtb43 +/+ spermatozoa. Heatmaps to the right in green show the affinity binding of MBP-ZBTB43-FL in unmethylated and methylated genomic DNA (TKO and SssI) centred at the sites of sperm DMRs. The sequencing results shown represent two independent biological replicates in b and d . e , ZBTB43 ChIP–seq peaks in 15.5 dpc prospermatogonia overlap with hypo-DMRs detected in Zbtb43 −/− sperm DNA. Venn diagrams. f , Model. Top: ZBTB43 binds Z-DNA in normal prospermatogonia. By removing Z-DNA, ZBTB43 creates an accessible substrate for DNMT3A and facilitates DNA methylation establishment at PPR-rich DNA regions. Bottom: Z-DNA is not removed in the Zbtb43 −/− prospermatogonia in the absence of ZBTB43, DNA methylation is not established at PPRs, and hypomethylated DMRs are found in mutant sperm. One prominent ZBTB43 region can induce large re-arrangements in mammalian mutation assays, and ZBTB43 protects from DSBs by directly binding to PPRs and removing mutagenic Z-DNA structures in the foetal germ cells.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Activity Assay, In Vitro, Binding Assay, Sequencing, Methylation, DNA Methylation Assay, Methylation Sequencing, In Vivo, ChIP-sequencing, Mutagenesis

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Venn diagram displays the relationship between ZBTB43 affinity peaks in fully methylated (SssI) genomic DNA and DMRs detected in Zbtb43 -/- sperm. ( b ) Venn diagram displays the relationship between ZBTB43 affinity peaks in fully unmethylated (TKO) genomic DNA and DMRs detected in Zbtb43 -/- sperm. ( c ) Venn diagram shows the relationship between predicted Z-DNA sites in the genome and DMRs detected in Zbtb43 -/- sperm. ( d ) Venn diagram shows the relationship between enriched Z-DNA sites in activated B-cells in the genome and DMRs detected in Zbtb43 -/- sperm.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Methylation

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Zbtb43 -/- pups out of the test cross are underrepresented at weaning. Venn diagram shows the distribution of genotypes at weaning from 55 pups out of 6 breeding pairs and 13 litters of the original Zbtb43 +/- heterozygous stock (HET x HET). The genotype distribution of live weanlings is also plotted from a test cross where the parents had been back-crossed 8-times in the JF1/Ms mouse strain (HET.JF1.N8 x HET.JF1.N8). These results were obtained from four breeding pairs, 19 litters and 70 pups. ( b ) Zbtb43 -/- pups are overrepresented among dead pups out of the HET x HET cross. Venn diagram shows the distribution of dead pups by genotype. We identified 6 Zbtb43 -/- dead pups out of 11 total dead pups from 2 litters of 1 breeding pair of the HET x HET initial cross by PCR genotyping. After backcrossing to C57BL/6N once, we identified 10 Zbtb43 -/- dead pups out 23 dead pups from 20 litters of 4 pairs of HET.B6 X Het B6. ( c ) Zbtb43 -/- dead pups (n=16) are overrepresented among all dead pups out of the HOMO x HET cross. Venn diagram shows the distribution of dead pups by genotype. ( d ) Partially penetrant lethality in the Zbtb43 mutant mouse line depends on parental genotype. The portion of newborn pups that died is depicted by bar graphs. The lethality phenotype is shown according to different parental crosses as indicated below each bar. The postnatal day of death (P0, P1, P2 and after P2) is shown by colored columns as coded to the right. Zbtb43 -/- (HOMO), and Zbtb43 +/- (HET) mice were obtained from the original JAX stock. Another set of mice were obtained after crossing to C57Bl/6N (B6) one time (HOMO B6, and HET B6). The mother is written first in each cross. The number of breeding pairs, litters, and pups born are provided under the chart. ( e ) The partial perinatal lethality phenotype of Zbtb43 -/- pups persists through generations (N1, N2, N3 and N4) in the HOMO x HOMO crosses. Total number of animals and litters in each generation are marked under the plot. ( f-g ) Zbtb43 -/- pups from Zbtb43 -/- parents exhibit reduced growth compared to Zbtb43 +/+ pups out of Zbtb43 +/+ parents. Weight of male ( f ) and female ( g ) pups is plotted at weeks 2, 3, 4, 5, 5, 6, and 8 after birth, and numbers of pups included in the measurements are given under the dot plots. Data are presented as mean values +/- SEM. Statistical analysis between genotypes at each age was done using multiple two-sided T-Tests. ( h-i ) Zbtb43 -/- pups exhibit reduced growth after weaning compared to their littermates. Growth of male ( h ) and female ( i ) pups out of HET x HET cross is depicted. Weight of WT, HET and HOMO pups is shown as coded by colors to the right and numbers of pups are given under the dot plots. Data are presented as mean values +/- SEM. Statistically significant difference is marked with P -value as determined using multiple two-sided T-Tests between pairs of genotypes at each age. ( j ) Parental genotype of Zbtb43 +/+ pups does not affect their growth. Growth curve of male and female WT pups is shown from 3 weeks to 8 weeks. Solid lines indicate the average weight of WT pups out of WT parents. Dashed lines indicate the average weight of WT pups out of the HET x HET cross. Data are presented as mean values +/- SEM. No statistically significant difference was found between crosses using multiple 2-sided T-Tests. ( k ) Parental genotype of male and female Zbtb43 -/- pups affects their growth after weaning. Growth curve of male and female HOMO pups is shown from 3 weeks to 8 weeks. Solid lines indicate the average weight of HOMO pups out of HOMO x HOMO cross. Dashed lines indicate the average weight of HOMO pups out of the HET x HET cross. Data are presented as mean values +/- SEM. Statistically significant differences between crosses at each age as obtained from multiple two-sided T-Tests are marked.

Article Snippet: His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl 2 ) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA.

Techniques: Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 1. Comparative Cellular Cytotoxicity Screen to identify TOP3B poisons. (A) A pair of isogenic HCT116 and HCT116-TOP3B-KO cells were constructed to express GFP and mCherry, respectively. Both cell lines were mixed at 1:1 ratio, seeded together, and allowed to attach for 48 h prior to treatment with drugs at indicated concentrations for an additional 72 h. On each cell culture plate, one column of dimethyl sulfoxide (DMSO)-treated samples served as control. At the end of drug treatment, the entire cell culture plate was imaged in multiple fluorescent channels to calculate the relative viability rates of the wild-type and TOP3B-KO cells, normalized to the DMSO-treated control wells on the same plate. The 4 potential outcomes are represented from left to right in the middle of the figure: 1) If TOP3B-KO cells show selective resistance, the compound potentially targets TOP3B; 2) if a compound is selectively toxic to TOP3B-KO cells, the compound potentially interferes with parallel pathways of TOP3B, so that in KO cells, inhibiting the parallel pathway leads to adverse effects; 3) if a compound shows no effect (same results as DMSO-treated samples), it does not interfere with any pathways related to TOP3B and is discarded; and 4) if a compound shows high toxicity to both HCT116 and TOP3B-KO cells, it is retested at lower concentrations. (B) Representative fluorescent microscopy images of a DMSO control and drug-treated samples are shown. After deconvoluting into individual fluorescent channels, the drug-treated sample was compared to the DMSO control sample in each fluorescent channel. The GFP channel conveys the effect of the drug on the HCT116 cells while the mCherry channel conveys the effect of the drug on TOP3B-KO cells. Each sample is normalized to the DMSO-treated control samples on the same plate.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Construct, Cell Culture, Control, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 3. Modified RADAR assay to demonstrate drug-induced cellular TOP3Bccs. (A) Schematic procedures of the modified RADAR assay. (B) Chemical structures of six hit compounds selected from the screen using modified RADAR assays. (C) HEK293 cells transiently transfected with Flag-tagged TOP3B (TOP3B OE) were treated with the indicated compounds at 100 µM for 1 h prior to analysis by modified RADAR. Anti-Flag antibody was used to probe the level of TOP3Bccs. The same samples probed with anti-dsDNA antibodies served as loading controls. The six compounds showed a spectrum of potency in inducing TOP3Bccs.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Modification, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 4. Band shift assays demonstrating the induction of DNA- and RNA-TOP3Bccs by NSC690634 and NSC96932 with recombinant TOP3B. (A) Simplified scheme of the band shift assay (11). A single-strand DNA or RNA oligo substrate (containing a short hairpin section) labeled on the 3′-end with a fluorophore is incubated with recombinant human TOP3B. A small population of the TOP3B forms TOP3Bccs with the DNA or RNA substrate. The resulting molecular weight of TOP3Bccs with the attached fluorophore is significantly higher than that of free DNA and RNA substrates, regardless of the positions of TOP3B binding sites. (B) The DNA-TOP3Bcc (Upper) and RNA-TOP3Bcc (Lower) are resolved from the free DNA and RNA on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and directly visualized by the fluorophores attached to the oligo constructs. The formation of DNA- and RNA-TOP3Bccs (100 to 180 kDa) is TOP3B- dependent and the TOP3Bccs were titrated with each of the six lead compounds to reach equilibrium. The effect of each compound is compared to the DMSO- treated control samples.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Electrophoretic Mobility Shift Assay, Recombinant, Labeling, Incubation, Molecular Weight, Binding Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Construct, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 5. NSC690634 and NSC96932 induce endogenous TOP3Bccs in different cell lines. (A) Representative modified RADAR assays showing that NSC690634 (100 µM) induces endogenous TOP3Bccs in a time-dependent manner in the indicated human cell lines, immunodetection with anti-TOP3B antibodies. (B) Modified RADAR assay showing that NSC690634 (4 h) induces endogenous TOP3Bccs in a dose-dependent manner in three human cell lines, immunodetection with anti-TOP3B antibodies. (C) Same as Fig. 5A, but with NSC96932. (D) Same as Fig. 5B, but with NSC96932 (1 h). (E) HEK293 cells transiently over-expressing TOP3B were treated with NSC690634 or NSC96932 (100 µM for 1 h) prior to analysis by in vivo complex of enzyme (ICE) bioassays. Ultracentrifugation of cell lysates on a CsCl gradient enabled separation of DNA and RNA species. The separated fractions were quantified and 1.5 µg of DNA or RNA were blotted on a slot blot for independent detection of DNA- and RNA-TOP3Bccs through immunodetection probed with anti-Flag antibodies. The upper row contained 1.5 µg of nontreated control DNA or RNA samples. Representative blot of 3 independent experiments is shown. (F) Genomic DNA of HCT116 and HCT116-TOP3B-KO cells with or without treatment with NSC690634 (100 µM, 4 h) was collected and blotted on nitrocellulose membrane before immunoprobing by S9.6 antibody (7). RNase H-treated samples serve as negative controls.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Modification, Immunodetection, Expressing, In Vivo, Dot Blot, Control, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 6. Structure–activity analysis of bisacridine analogs of NSC690634 demonstrates that linker length is critical for inducing TOP3Bccs. (A) Chemical structures of a series of bisacridines. The length of the carbon linker between the two acridine moieties increases from left to right. (B) HEK293 cells transiently over-expressing TOP3B (TOP3B OE) were treated with DMSO (control) or with the indicated bisacridines (100 µM for 1 h) prior to analysis by modified RADAR and probed with anti-Flag antibody. The same samples probed with anti-dsDNA antibody served as loading controls. Only NSC690634 showed a strong induction of TOP3Bccs while all the other structurally related compounds with different linker lengths failed to induce cellular TOP3Bccs. (C) In vitro band shift assays demonstrate that linker length is critical for inducing TOP3Bccs by the bisacridine NSC690634. Compounds without linkers did not trap TOP3B, whereas NSC690634 (3-carbon linker) strongly enhanced TOP3Bccs with RNA. Bisacridine compounds with longer carbon linkers not only failed to induce TOP3Bcc but they also destabilized TOP3Bccs with both DNA and RNA.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Activity Assay, Expressing, Control, Modification, In Vitro, Electrophoretic Mobility Shift Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 7. Structure activity analysis of thiacyanine analogs of NSC96932 demonstrates that the linker length is critical for inducing TOP3Bccs. (A) Chemical structures of a series of thiacyanine analogs. The length of the carbon linker between the two indole ring moieties increases from Left to Right. (B) HEK293 cells transiently over-expressing TOP3B (TOP3B OE) were treated with the indicated thiacyanine analogs (100 µM for 1 h) prior to analysis by modified RADAR and probed with anti-Flag antibody. The same samples probed with anti-dsDNA antibody served as loading controls. Only the thiacyanine with 3-carbon linkers (NSC96932, NSC356711, and NSC93472) induced endogenous TOP3Bccs while the compounds with longer carbon-linker lengths failed to induce cellular TOP3Bccs.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Activity Assay, Expressing, Modification

Journal: Cancers

Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells

doi: 10.3390/cancers10100371

Figure Lengend Snippet: DNA methylation impedes PR binding to progesterone-responsive elements (PREs). ( a ) Screen shot from the UCSC genome browser showing the CpG island (CpG 89) at the ESR1 promoter and the positions of the canonical PREs containing a CpG (blue line) and six half-palindromic PREs with one or two neighboring CpGs (red lines). ( b , c ) Electrophoretic-mobility shift assay using the indicated amount of purified human PR to capture the PRE with no CpG (ACAGTTTGT; no CpG), one methylated (MetCpG) or unmethylated CpG (UnmetCpG) (ACGGTTTGT) ( b ); two methylated (MetCpGs) or two unmethylated CpGs (UnmetCpGs) (ACGGTTCGT) ( c ). Quantification of the percentage of PR binding to different probes is shown in the lower part of the gel images. Error bars represent the SD of three independent experiments. * p less than or equal to 0.05, ** p less than or equal to 0.01, unpaired two-tailed Student’s t -test. ( d ) A double-stranded oligonucleotide probe with no CpG (ACAGTTTGT) was incubated with 2.4 μg of purified human PR and analyzed by PAGE either in the absence (–) or presence (+) of 100-fold excess of unlabeled oligonucleotides containing two unmethylated (UNMET) or two methylated (MET) CpGs (ACGGTTCGT). Error bars represent the SD of three independent experiments. *** p less than or equal to 0.005, unpaired two-tailed Student’s t -test. The dashed grey line indicates that a lane between the two samples was removed.

Article Snippet: Chromatin was sheared by sonication and incubated 16 h with 5 μg of antibody against progesterone receptor (PR, Santa Cruz Biotechnology, Dallas, TX, USA, sc-7208) or rabbit IgG (Cell Signaling, Danvers, MA, USA, #2729s).

Techniques: DNA Methylation Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Methylation, Two Tailed Test, Incubation