Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 and G4 are present at many gene promoters and regulate metastasis-associated genes in TNBC cells. ( A ) Volcano plot of RNA-seq data showing significantly down-regulated or up-regulated genes upon APE1 KO in MDA-MB-231 cells. Red and blue points indicate differentially increased and decreased genes, respectively, under thresholds of adjusted P -value <0.05 and log2 fold change > |1|. ( B ) Quantification of gene expression by qRT-PCR in WT and APE1-KO TNBC cells. ( C ) Analysis of genome-wide APE1 binding relative to genomic features in MDA-MB-231 cells. ( D ) Heatmaps of APE1 and H3K27ac ChIP-seq signal at peaks (summits) sorted by H3K27ac occupancy in MDA-MB-231 cells. ( E ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the CXCL1 gene promoter in MDA-MB-231 cells (upper panel). Potential G4-forming sequence in the CXCL1 promoter region (−145 bp to −167 bp upstream of the TSS) that overlaps with APE1’s peak is shown (lower panel). ( F ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the VEGFA gene promoter region in MDA-MB-231 cells (upper panel). Two potential G4-forming sequences VEGFA G4-#1 (−68 bp to −88 bp upstream of the TSS) and VEGFA G4-#2 (−387 bp to −406 bp upstream of the TSS) in the VEGFA promoter are shown (lower panel). ( G ) CD spectroscopy of CXCL1 G4-forming oligo showing formation of a hybrid (signatures characteristic maxima ∼265 nm and ∼295 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. CD spectroscopy of VEGFA promoter G4-forming oligo showing formation of parallel (signatures characteristic maxima ∼265 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. The y -axis indicates the ellipticity signal expressed in millidegrees. ( H ) Promoter-directed ChIP assay using G4-specific antibody BG4 in TNBC cells shows enrichment of folded G4 structures at CXCL1 and VEGFA promoter regions. Fold enrichment with G4-targeting antibody versus control IgG was calculated after input normalization. ( I ) Promoter-directed ChIP assay using APE1 antibody in TNBC cells shows enrichment of APE1 at CXCL1 promoter and VEGFA promoter G4 regions. Fold enrichment with anti-APE1 antibody versus control IgG was calculated after input normalization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Genome Wide, Binding Assay, ChIP-sequencing, Sequencing, Circular Dichroism, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 binds to G4 structures in vitro and overlaps with G4 in cells. ( A ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), random double-stranded DNA oligo (DSD), and non-G4-forming random single-stranded oligo (Non-G4-random) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( B ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1- or G4 #2-forming oligo, non-G4-forming single-stranded (Non-G4-random), or random DSD were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( C ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), complementary C-rich single-stranded DNA oligo (CXCL1-G4 complement C-rich SSD), CXCL1 G4 double-stranded DNA oligo (CXCL1-G4 DSD), or Non-G4-random labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed using APE1 antibody. Non-linear regression was used to analyze the data. ( D ) Representative images of tghe PLA show APE1 and G4 proximity (distance ≤ 40nm) in the nucleus in WT and APE1-KO TNBC cells. G4–APE1 PLA foci were visualized by confocal microscopy imaging (magnification: ×63). No antibody, anti-APE1 alone, G4 antibody alone, or BCL2 antibody, served as negative controls.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: In Vitro, Labeling, Incubation, Recombinant, Confocal Microscopy, Imaging

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 loops and APE1’s N-terminus are crucial for APE1–G4 interaction. ( A ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1 oligo (forming parallel G4), CXCL1 G4 oligo (forming hybrid G4), or telomeric G4 oligo (forming antiparallel G4 in Na + buffer) were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( B ) VEGFA G4-#1 WT (VEGFA-G4-WT) or loop-shortened G4 oligos (VEGFA-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) CXCL1 WT (CXCL1-G4-WT) or loop-shortened G4 oligos (CXCL1-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( D and E ) ELISAs with 50 nM biotin-labeled CXCL1 -G4 oligo or VEGFA G4 #1 oligo were performed with increasing concentrations of recombinant WT APE1 or APE1 with the N-terminal 1–42 amino acids truncated (DeltaN42), respectively. Data were analyzed by non-linear regression.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: Labeling, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 is crucial to recruit APE1 to the CXCL1 gene promoter. ( A ) Schematic overview of CRISPR/Cas9-mediated generation of knock-in mutations in the CXCL1 promoter G4 sequence; the WT CXCL1 G4 sequence and mutated G4 sequences are shown (left panel). Sanger sequencing confirmed the homologous mutations (in both alleles) in CXCL1 G4 sequence ( CXCL1 -G4 Mut) compared with the CXCL1 -G4 WT sequence (right panel). ( B ) Enrichment of folded G4 structure in the CXCL1 mutated G4 ( CXCL1 -G4 Mut) promoter versus the CXCL1 WT G4 promoter region in MDA-MB-231 and BT-549 cells was examined by promoter-directed ChIP assay with G4-specific antibody. ( C ) Promoter-directed ChIP assay with APE1 antibody in TNBC cells shows enrichment of APE1 in the CXCL1 -G4 WT promoter region and the CXCL1 -G4 Mut promoter. ( D ) Quantitation of CXCL1 expression in TNBC cells containing the CXCL1 -G4 WT and CXCL1 -G4 Mut promoter by qRT-PCR. * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: CRISPR, Knock-In, Sequencing, Quantitation Assay, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: The APE1–G4 axis promotes TF binding at promoters. ( A and B ) Promoter-directed ChIP assay shows enrichment of Snail1 at the CXCL1 promoter(A) and of c-Jun at the VEGFA promoter (B) in WT and APE1-KO cells. ( C ) Promoter-directed ChIP assay shows enrichment of Snail1 in CXCL1 -G4 WT and CXCL1 -G4 Mut promoter-containing cells. ( D ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), APE1 Ref-1 function-defective mutant (KO-CS), or APE1 endonuclease-defective mutant (KO-ED) under a Dox-inducible promoter were treated with 2 µg ml −1 Dox to induce the expression of APE1. Then the expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ( E ) Representative images of PLAs of APE1 and G4 co-localization in APE1-KO MDA-MB-231 cells expressing WT-APE1 (WT) or the APE1 N-terminal deletion mutant (DeltaN42) under treatment with 2 µg ml −1 Dox (left panel); average numbers of PLA foci of 30 cells were quantitated (right panel). ( F ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), or the APE1 N-terminal deletion mutant (KO-DeltaN42) under treatment with 2 µg ml −1 Dox. The expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: Binding Assay, Expressing, Control, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 ligands blocks G4–APE1 interaction. ( A ) MDA-MB-231 cells treated with either vehicle or the G4-stabilizing ligand TMPyP4 (150 µM) for 24 h and then immunostained with G4-specific antibody and visualized by confocal microscopy. ( B ) CXCL1 -G4 or VEGFA -G4 oligos labeled with 50 nM biotin, which were attached to a streptavidin-conjugated plate, were incubated with a saturating dose (64 nM) of APE1 protein and 50 mM KCl, titrated with increasing concentrations of TMPyP4, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle or TMPyP4 (150 µM) (left panel); average numbers of PLA foci of ~25 cells were quantitated (right panel). ( D ) Promoter-directed ChIP assay shows enrichment of APE1 versus IgG at CXCL1 G4 and VEGFA G4 promoter regions following 150 µM TMPyP4 treatment. ( E ) Expression levels of genes involved in migration in TNBC cells after treatment with vehicle or 150 µM TMPyP4 for 24 h, determined by qRT-PCR. ( F ) CXCL1 -G4 or VEGFA -G4-#1 oligos labeled with 50 nM biotin were incubated with 64 nM APE1 protein, titrated with increasing concentrations of PDS or PhenDC3, respectively, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( G ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle, PDS (10 μM), or PhenDC3 (20 μM) (left panel); PLA foci of ~30 cells were quantitated in each group (right panel). * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: Confocal Microscopy, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Migration, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: Disruption of the APE1–G4 axis inhibits TNBC tumor growth and lung metastasis in vivo . ( A ) Representative bioluminescence IVIS images of a nude mouse showing tumor burdens at days 21, 35, 52, and 63 after orthotopic implantation of MDA-MB-231 APE1-WT (mice n = 5) and APE1-KO cells (mice n = 4) in the mammary fat pad of nude mice. A different bioluminescence intensity scale was used to show the tumor burden on the indicated days. ( B ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing APE1-WT (mice n = 5) or APE1-KO (mice n = 4) tumors shown in (A). ( C ) Bioluminescence imaging of lungs harvested from WT and APE1-KO TNBC tumor-bearing mice as shown in(A), exhibiting tumor metastases to the lungs. ( D ) Quantitation of bioluminescence fluxes of lung metastatic tumors in each WT and APE1-KO group as shown in (C). ( E ) Representative bioluminescence imaging of a nude mouse tumor burden at days 21, 36, 47, and 62 after orthotopic implantation of MDA-MB-231 CXCL1 -G4 WT- ( n = 5) and CXCL1 -G4 Mut- ( n = 5) containing cells. Mice ( n = 5) bearing MDA-MB-231 CXCL1 -G4 WT tumors were treated with 10 mg kg −1 TMPyP4 twice weekly. The IVIS images were taken on the indicated days. (F ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing CXCL1 -G4 Mut ( n = 5) or CXCL1 -G4 WT ( n = 5) tumors treated with or without TMPyP4, as shown in (E).( G ) CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated primary tumors were allowed to grow for different time periods until they reached 1.5 cm in diameter, mice were then euthanized, and lung tissues were harvested. Bioluminescence imaging of lungs harvested from CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated TNBC tumor-bearing mice as shown in(E). ( H ) Quantification of bioluminescence intensity of lung tissues in MDA-MB-231 CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated groups as shown in (G).

Article Snippet: Primary antibodies used included: APE1 (Novus, Cat# NB100-101), Snail (Cell Signaling Technology, Cat# 3879), c-Jun (Cell Signaling Technology, Cat# 9165), and HSC70 (Santa Cruz Biotechnology, Cat# sc-7298).

Techniques: Disruption, In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 and G4 are present at many gene promoters and regulate metastasis-associated genes in TNBC cells. ( A ) Volcano plot of RNA-seq data showing significantly down-regulated or up-regulated genes upon APE1 KO in MDA-MB-231 cells. Red and blue points indicate differentially increased and decreased genes, respectively, under thresholds of adjusted P -value <0.05 and log2 fold change > |1|. ( B ) Quantification of gene expression by qRT-PCR in WT and APE1-KO TNBC cells. ( C ) Analysis of genome-wide APE1 binding relative to genomic features in MDA-MB-231 cells. ( D ) Heatmaps of APE1 and H3K27ac ChIP-seq signal at peaks (summits) sorted by H3K27ac occupancy in MDA-MB-231 cells. ( E ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the CXCL1 gene promoter in MDA-MB-231 cells (upper panel). Potential G4-forming sequence in the CXCL1 promoter region (−145 bp to −167 bp upstream of the TSS) that overlaps with APE1’s peak is shown (lower panel). ( F ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the VEGFA gene promoter region in MDA-MB-231 cells (upper panel). Two potential G4-forming sequences VEGFA G4-#1 (−68 bp to −88 bp upstream of the TSS) and VEGFA G4-#2 (−387 bp to −406 bp upstream of the TSS) in the VEGFA promoter are shown (lower panel). ( G ) CD spectroscopy of CXCL1 G4-forming oligo showing formation of a hybrid (signatures characteristic maxima ∼265 nm and ∼295 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. CD spectroscopy of VEGFA promoter G4-forming oligo showing formation of parallel (signatures characteristic maxima ∼265 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. The y -axis indicates the ellipticity signal expressed in millidegrees. ( H ) Promoter-directed ChIP assay using G4-specific antibody BG4 in TNBC cells shows enrichment of folded G4 structures at CXCL1 and VEGFA promoter regions. Fold enrichment with G4-targeting antibody versus control IgG was calculated after input normalization. ( I ) Promoter-directed ChIP assay using APE1 antibody in TNBC cells shows enrichment of APE1 at CXCL1 promoter and VEGFA promoter G4 regions. Fold enrichment with anti-APE1 antibody versus control IgG was calculated after input normalization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Genome Wide, Binding Assay, ChIP-sequencing, Sequencing, Circular Dichroism, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 binds to G4 structures in vitro and overlaps with G4 in cells. ( A ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), random double-stranded DNA oligo (DSD), and non-G4-forming random single-stranded oligo (Non-G4-random) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( B ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1- or G4 #2-forming oligo, non-G4-forming single-stranded (Non-G4-random), or random DSD were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( C ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), complementary C-rich single-stranded DNA oligo (CXCL1-G4 complement C-rich SSD), CXCL1 G4 double-stranded DNA oligo (CXCL1-G4 DSD), or Non-G4-random labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed using APE1 antibody. Non-linear regression was used to analyze the data. ( D ) Representative images of tghe PLA show APE1 and G4 proximity (distance ≤ 40nm) in the nucleus in WT and APE1-KO TNBC cells. G4–APE1 PLA foci were visualized by confocal microscopy imaging (magnification: ×63). No antibody, anti-APE1 alone, G4 antibody alone, or BCL2 antibody, served as negative controls.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: In Vitro, Labeling, Incubation, Recombinant, Confocal Microscopy, Imaging

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 loops and APE1’s N-terminus are crucial for APE1–G4 interaction. ( A ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1 oligo (forming parallel G4), CXCL1 G4 oligo (forming hybrid G4), or telomeric G4 oligo (forming antiparallel G4 in Na + buffer) were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( B ) VEGFA G4-#1 WT (VEGFA-G4-WT) or loop-shortened G4 oligos (VEGFA-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) CXCL1 WT (CXCL1-G4-WT) or loop-shortened G4 oligos (CXCL1-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( D and E ) ELISAs with 50 nM biotin-labeled CXCL1 -G4 oligo or VEGFA G4 #1 oligo were performed with increasing concentrations of recombinant WT APE1 or APE1 with the N-terminal 1–42 amino acids truncated (DeltaN42), respectively. Data were analyzed by non-linear regression.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: Labeling, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 is crucial to recruit APE1 to the CXCL1 gene promoter. ( A ) Schematic overview of CRISPR/Cas9-mediated generation of knock-in mutations in the CXCL1 promoter G4 sequence; the WT CXCL1 G4 sequence and mutated G4 sequences are shown (left panel). Sanger sequencing confirmed the homologous mutations (in both alleles) in CXCL1 G4 sequence ( CXCL1 -G4 Mut) compared with the CXCL1 -G4 WT sequence (right panel). ( B ) Enrichment of folded G4 structure in the CXCL1 mutated G4 ( CXCL1 -G4 Mut) promoter versus the CXCL1 WT G4 promoter region in MDA-MB-231 and BT-549 cells was examined by promoter-directed ChIP assay with G4-specific antibody. ( C ) Promoter-directed ChIP assay with APE1 antibody in TNBC cells shows enrichment of APE1 in the CXCL1 -G4 WT promoter region and the CXCL1 -G4 Mut promoter. ( D ) Quantitation of CXCL1 expression in TNBC cells containing the CXCL1 -G4 WT and CXCL1 -G4 Mut promoter by qRT-PCR. * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: CRISPR, Knock-In, Sequencing, Quantitation Assay, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: The APE1–G4 axis promotes TF binding at promoters. ( A and B ) Promoter-directed ChIP assay shows enrichment of Snail1 at the CXCL1 promoter(A) and of c-Jun at the VEGFA promoter (B) in WT and APE1-KO cells. ( C ) Promoter-directed ChIP assay shows enrichment of Snail1 in CXCL1 -G4 WT and CXCL1 -G4 Mut promoter-containing cells. ( D ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), APE1 Ref-1 function-defective mutant (KO-CS), or APE1 endonuclease-defective mutant (KO-ED) under a Dox-inducible promoter were treated with 2 µg ml −1 Dox to induce the expression of APE1. Then the expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ( E ) Representative images of PLAs of APE1 and G4 co-localization in APE1-KO MDA-MB-231 cells expressing WT-APE1 (WT) or the APE1 N-terminal deletion mutant (DeltaN42) under treatment with 2 µg ml −1 Dox (left panel); average numbers of PLA foci of 30 cells were quantitated (right panel). ( F ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), or the APE1 N-terminal deletion mutant (KO-DeltaN42) under treatment with 2 µg ml −1 Dox. The expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: Binding Assay, Expressing, Control, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 ligands blocks G4–APE1 interaction. ( A ) MDA-MB-231 cells treated with either vehicle or the G4-stabilizing ligand TMPyP4 (150 µM) for 24 h and then immunostained with G4-specific antibody and visualized by confocal microscopy. ( B ) CXCL1 -G4 or VEGFA -G4 oligos labeled with 50 nM biotin, which were attached to a streptavidin-conjugated plate, were incubated with a saturating dose (64 nM) of APE1 protein and 50 mM KCl, titrated with increasing concentrations of TMPyP4, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle or TMPyP4 (150 µM) (left panel); average numbers of PLA foci of ~25 cells were quantitated (right panel). ( D ) Promoter-directed ChIP assay shows enrichment of APE1 versus IgG at CXCL1 G4 and VEGFA G4 promoter regions following 150 µM TMPyP4 treatment. ( E ) Expression levels of genes involved in migration in TNBC cells after treatment with vehicle or 150 µM TMPyP4 for 24 h, determined by qRT-PCR. ( F ) CXCL1 -G4 or VEGFA -G4-#1 oligos labeled with 50 nM biotin were incubated with 64 nM APE1 protein, titrated with increasing concentrations of PDS or PhenDC3, respectively, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( G ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle, PDS (10 μM), or PhenDC3 (20 μM) (left panel); PLA foci of ~30 cells were quantitated in each group (right panel). * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: Confocal Microscopy, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Migration, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: Disruption of the APE1–G4 axis inhibits TNBC tumor growth and lung metastasis in vivo . ( A ) Representative bioluminescence IVIS images of a nude mouse showing tumor burdens at days 21, 35, 52, and 63 after orthotopic implantation of MDA-MB-231 APE1-WT (mice n = 5) and APE1-KO cells (mice n = 4) in the mammary fat pad of nude mice. A different bioluminescence intensity scale was used to show the tumor burden on the indicated days. ( B ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing APE1-WT (mice n = 5) or APE1-KO (mice n = 4) tumors shown in (A). ( C ) Bioluminescence imaging of lungs harvested from WT and APE1-KO TNBC tumor-bearing mice as shown in(A), exhibiting tumor metastases to the lungs. ( D ) Quantitation of bioluminescence fluxes of lung metastatic tumors in each WT and APE1-KO group as shown in (C). ( E ) Representative bioluminescence imaging of a nude mouse tumor burden at days 21, 36, 47, and 62 after orthotopic implantation of MDA-MB-231 CXCL1 -G4 WT- ( n = 5) and CXCL1 -G4 Mut- ( n = 5) containing cells. Mice ( n = 5) bearing MDA-MB-231 CXCL1 -G4 WT tumors were treated with 10 mg kg −1 TMPyP4 twice weekly. The IVIS images were taken on the indicated days. (F ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing CXCL1 -G4 Mut ( n = 5) or CXCL1 -G4 WT ( n = 5) tumors treated with or without TMPyP4, as shown in (E).( G ) CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated primary tumors were allowed to grow for different time periods until they reached 1.5 cm in diameter, mice were then euthanized, and lung tissues were harvested. Bioluminescence imaging of lungs harvested from CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated TNBC tumor-bearing mice as shown in(E). ( H ) Quantification of bioluminescence intensity of lung tissues in MDA-MB-231 CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated groups as shown in (G).

Article Snippet: After pre-clearing, immunoprecipitations were conducted overnight at 4°C with antibodies against APE1 (Novus, Cat# NB100-101) or acetylated APE1 [ ], Snail (Cell Signaling Technology, Cat# 3879), or c-Jun (Cell Signaling Technology, Cat# 9165).

Techniques: Disruption, In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 and G4 are present at many gene promoters and regulate metastasis-associated genes in TNBC cells. ( A ) Volcano plot of RNA-seq data showing significantly down-regulated or up-regulated genes upon APE1 KO in MDA-MB-231 cells. Red and blue points indicate differentially increased and decreased genes, respectively, under thresholds of adjusted P -value <0.05 and log2 fold change > |1|. ( B ) Quantification of gene expression by qRT-PCR in WT and APE1-KO TNBC cells. ( C ) Analysis of genome-wide APE1 binding relative to genomic features in MDA-MB-231 cells. ( D ) Heatmaps of APE1 and H3K27ac ChIP-seq signal at peaks (summits) sorted by H3K27ac occupancy in MDA-MB-231 cells. ( E ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the CXCL1 gene promoter in MDA-MB-231 cells (upper panel). Potential G4-forming sequence in the CXCL1 promoter region (−145 bp to −167 bp upstream of the TSS) that overlaps with APE1’s peak is shown (lower panel). ( F ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the VEGFA gene promoter region in MDA-MB-231 cells (upper panel). Two potential G4-forming sequences VEGFA G4-#1 (−68 bp to −88 bp upstream of the TSS) and VEGFA G4-#2 (−387 bp to −406 bp upstream of the TSS) in the VEGFA promoter are shown (lower panel). ( G ) CD spectroscopy of CXCL1 G4-forming oligo showing formation of a hybrid (signatures characteristic maxima ∼265 nm and ∼295 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. CD spectroscopy of VEGFA promoter G4-forming oligo showing formation of parallel (signatures characteristic maxima ∼265 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. The y -axis indicates the ellipticity signal expressed in millidegrees. ( H ) Promoter-directed ChIP assay using G4-specific antibody BG4 in TNBC cells shows enrichment of folded G4 structures at CXCL1 and VEGFA promoter regions. Fold enrichment with G4-targeting antibody versus control IgG was calculated after input normalization. ( I ) Promoter-directed ChIP assay using APE1 antibody in TNBC cells shows enrichment of APE1 at CXCL1 promoter and VEGFA promoter G4 regions. Fold enrichment with anti-APE1 antibody versus control IgG was calculated after input normalization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Genome Wide, Binding Assay, ChIP-sequencing, Sequencing, Circular Dichroism, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 binds to G4 structures in vitro and overlaps with G4 in cells. ( A ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), random double-stranded DNA oligo (DSD), and non-G4-forming random single-stranded oligo (Non-G4-random) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( B ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1- or G4 #2-forming oligo, non-G4-forming single-stranded (Non-G4-random), or random DSD were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( C ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), complementary C-rich single-stranded DNA oligo (CXCL1-G4 complement C-rich SSD), CXCL1 G4 double-stranded DNA oligo (CXCL1-G4 DSD), or Non-G4-random labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed using APE1 antibody. Non-linear regression was used to analyze the data. ( D ) Representative images of tghe PLA show APE1 and G4 proximity (distance ≤ 40nm) in the nucleus in WT and APE1-KO TNBC cells. G4–APE1 PLA foci were visualized by confocal microscopy imaging (magnification: ×63). No antibody, anti-APE1 alone, G4 antibody alone, or BCL2 antibody, served as negative controls.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: In Vitro, Labeling, Incubation, Recombinant, Confocal Microscopy, Imaging

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 loops and APE1’s N-terminus are crucial for APE1–G4 interaction. ( A ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1 oligo (forming parallel G4), CXCL1 G4 oligo (forming hybrid G4), or telomeric G4 oligo (forming antiparallel G4 in Na + buffer) were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( B ) VEGFA G4-#1 WT (VEGFA-G4-WT) or loop-shortened G4 oligos (VEGFA-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) CXCL1 WT (CXCL1-G4-WT) or loop-shortened G4 oligos (CXCL1-G4-Short loops) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( D and E ) ELISAs with 50 nM biotin-labeled CXCL1 -G4 oligo or VEGFA G4 #1 oligo were performed with increasing concentrations of recombinant WT APE1 or APE1 with the N-terminal 1–42 amino acids truncated (DeltaN42), respectively. Data were analyzed by non-linear regression.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: Labeling, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 is crucial to recruit APE1 to the CXCL1 gene promoter. ( A ) Schematic overview of CRISPR/Cas9-mediated generation of knock-in mutations in the CXCL1 promoter G4 sequence; the WT CXCL1 G4 sequence and mutated G4 sequences are shown (left panel). Sanger sequencing confirmed the homologous mutations (in both alleles) in CXCL1 G4 sequence ( CXCL1 -G4 Mut) compared with the CXCL1 -G4 WT sequence (right panel). ( B ) Enrichment of folded G4 structure in the CXCL1 mutated G4 ( CXCL1 -G4 Mut) promoter versus the CXCL1 WT G4 promoter region in MDA-MB-231 and BT-549 cells was examined by promoter-directed ChIP assay with G4-specific antibody. ( C ) Promoter-directed ChIP assay with APE1 antibody in TNBC cells shows enrichment of APE1 in the CXCL1 -G4 WT promoter region and the CXCL1 -G4 Mut promoter. ( D ) Quantitation of CXCL1 expression in TNBC cells containing the CXCL1 -G4 WT and CXCL1 -G4 Mut promoter by qRT-PCR. * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: CRISPR, Knock-In, Sequencing, Quantitation Assay, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: The APE1–G4 axis promotes TF binding at promoters. ( A and B ) Promoter-directed ChIP assay shows enrichment of Snail1 at the CXCL1 promoter(A) and of c-Jun at the VEGFA promoter (B) in WT and APE1-KO cells. ( C ) Promoter-directed ChIP assay shows enrichment of Snail1 in CXCL1 -G4 WT and CXCL1 -G4 Mut promoter-containing cells. ( D ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), APE1 Ref-1 function-defective mutant (KO-CS), or APE1 endonuclease-defective mutant (KO-ED) under a Dox-inducible promoter were treated with 2 µg ml −1 Dox to induce the expression of APE1. Then the expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ( E ) Representative images of PLAs of APE1 and G4 co-localization in APE1-KO MDA-MB-231 cells expressing WT-APE1 (WT) or the APE1 N-terminal deletion mutant (DeltaN42) under treatment with 2 µg ml −1 Dox (left panel); average numbers of PLA foci of 30 cells were quantitated (right panel). ( F ) MDA-MB-231 APE1-KO cells expressing control vector (KO-Vec), WT-APE1 (KO-WT), or the APE1 N-terminal deletion mutant (KO-DeltaN42) under treatment with 2 µg ml −1 Dox. The expression levels of CXCL1 and VEGFA were quantitated in each group of cells by qRT-PCR. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: Binding Assay, Expressing, Control, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: G4 ligands blocks G4–APE1 interaction. ( A ) MDA-MB-231 cells treated with either vehicle or the G4-stabilizing ligand TMPyP4 (150 µM) for 24 h and then immunostained with G4-specific antibody and visualized by confocal microscopy. ( B ) CXCL1 -G4 or VEGFA -G4 oligos labeled with 50 nM biotin, which were attached to a streptavidin-conjugated plate, were incubated with a saturating dose (64 nM) of APE1 protein and 50 mM KCl, titrated with increasing concentrations of TMPyP4, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( C ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle or TMPyP4 (150 µM) (left panel); average numbers of PLA foci of ~25 cells were quantitated (right panel). ( D ) Promoter-directed ChIP assay shows enrichment of APE1 versus IgG at CXCL1 G4 and VEGFA G4 promoter regions following 150 µM TMPyP4 treatment. ( E ) Expression levels of genes involved in migration in TNBC cells after treatment with vehicle or 150 µM TMPyP4 for 24 h, determined by qRT-PCR. ( F ) CXCL1 -G4 or VEGFA -G4-#1 oligos labeled with 50 nM biotin were incubated with 64 nM APE1 protein, titrated with increasing concentrations of PDS or PhenDC3, respectively, and then an ELISA was performed with APE1 antibody. Non-linear regression was used to analyze the data. ( G ) Representative images of PLAs of APE1 and G4 co-localization in TNBC cells after treatment with vehicle, PDS (10 μM), or PhenDC3 (20 μM) (left panel); PLA foci of ~30 cells were quantitated in each group (right panel). * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: Confocal Microscopy, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Migration, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: Disruption of the APE1–G4 axis inhibits TNBC tumor growth and lung metastasis in vivo . ( A ) Representative bioluminescence IVIS images of a nude mouse showing tumor burdens at days 21, 35, 52, and 63 after orthotopic implantation of MDA-MB-231 APE1-WT (mice n = 5) and APE1-KO cells (mice n = 4) in the mammary fat pad of nude mice. A different bioluminescence intensity scale was used to show the tumor burden on the indicated days. ( B ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing APE1-WT (mice n = 5) or APE1-KO (mice n = 4) tumors shown in (A). ( C ) Bioluminescence imaging of lungs harvested from WT and APE1-KO TNBC tumor-bearing mice as shown in(A), exhibiting tumor metastases to the lungs. ( D ) Quantitation of bioluminescence fluxes of lung metastatic tumors in each WT and APE1-KO group as shown in (C). ( E ) Representative bioluminescence imaging of a nude mouse tumor burden at days 21, 36, 47, and 62 after orthotopic implantation of MDA-MB-231 CXCL1 -G4 WT- ( n = 5) and CXCL1 -G4 Mut- ( n = 5) containing cells. Mice ( n = 5) bearing MDA-MB-231 CXCL1 -G4 WT tumors were treated with 10 mg kg −1 TMPyP4 twice weekly. The IVIS images were taken on the indicated days. (F ) Line chart of luciferase bioluminescence intensity showing tumor burden over time in mice bearing CXCL1 -G4 Mut ( n = 5) or CXCL1 -G4 WT ( n = 5) tumors treated with or without TMPyP4, as shown in (E).( G ) CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated primary tumors were allowed to grow for different time periods until they reached 1.5 cm in diameter, mice were then euthanized, and lung tissues were harvested. Bioluminescence imaging of lungs harvested from CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated TNBC tumor-bearing mice as shown in(E). ( H ) Quantification of bioluminescence intensity of lung tissues in MDA-MB-231 CXCL1 -G4 WT, CXCL1 -G4 Mut, and TMPyP4-treated groups as shown in (G).

Article Snippet: Other primary antibodies include APE1 (Novus, Cat# NB100-101).

Techniques: Disruption, In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 and G4 are present at many gene promoters and regulate metastasis-associated genes in TNBC cells. ( A ) Volcano plot of RNA-seq data showing significantly down-regulated or up-regulated genes upon APE1 KO in MDA-MB-231 cells. Red and blue points indicate differentially increased and decreased genes, respectively, under thresholds of adjusted P -value <0.05 and log2 fold change > |1|. ( B ) Quantification of gene expression by qRT-PCR in WT and APE1-KO TNBC cells. ( C ) Analysis of genome-wide APE1 binding relative to genomic features in MDA-MB-231 cells. ( D ) Heatmaps of APE1 and H3K27ac ChIP-seq signal at peaks (summits) sorted by H3K27ac occupancy in MDA-MB-231 cells. ( E ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the CXCL1 gene promoter in MDA-MB-231 cells (upper panel). Potential G4-forming sequence in the CXCL1 promoter region (−145 bp to −167 bp upstream of the TSS) that overlaps with APE1’s peak is shown (lower panel). ( F ) Genome browser screenshot illustrating the overlap between APE1 and H3K27ac peaks in the VEGFA gene promoter region in MDA-MB-231 cells (upper panel). Two potential G4-forming sequences VEGFA G4-#1 (−68 bp to −88 bp upstream of the TSS) and VEGFA G4-#2 (−387 bp to −406 bp upstream of the TSS) in the VEGFA promoter are shown (lower panel). ( G ) CD spectroscopy of CXCL1 G4-forming oligo showing formation of a hybrid (signatures characteristic maxima ∼265 nm and ∼295 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. CD spectroscopy of VEGFA promoter G4-forming oligo showing formation of parallel (signatures characteristic maxima ∼265 nm, minima ∼245 nm) G4 structure in vitro in the presence of 50 mM KCl. The y -axis indicates the ellipticity signal expressed in millidegrees. ( H ) Promoter-directed ChIP assay using G4-specific antibody BG4 in TNBC cells shows enrichment of folded G4 structures at CXCL1 and VEGFA promoter regions. Fold enrichment with G4-targeting antibody versus control IgG was calculated after input normalization. ( I ) Promoter-directed ChIP assay using APE1 antibody in TNBC cells shows enrichment of APE1 at CXCL1 promoter and VEGFA promoter G4 regions. Fold enrichment with anti-APE1 antibody versus control IgG was calculated after input normalization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Student’s t -test.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies: anti-APE1 (Novus, Cat# NB100-101), anti-Ki67 (Abcam, Cat# ab15580), anti-mouse IgG (Santa Cruz, Cat# sc-2025), and anti-rabbit IgG (Cell Signaling Technology, Cat# 2729).

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Genome Wide, Binding Assay, ChIP-sequencing, Sequencing, Circular Dichroism, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: Endogenous promoter G-quadruplexes scaffold apurinic/apyrimidinic endonuclease (APE1) to drive gene expression

doi: 10.1093/nar/gkag284

Figure Lengend Snippet: APE1 binds to G4 structures in vitro and overlaps with G4 in cells. ( A ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), random double-stranded DNA oligo (DSD), and non-G4-forming random single-stranded oligo (Non-G4-random) labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed with APE1 antibody. Non-linear regression was used to analyze the data. ( B ) ELISAs with 50 nM biotin-labeled VEGFA G4 #1- or G4 #2-forming oligo, non-G4-forming single-stranded (Non-G4-random), or random DSD were performed with increasing concentrations of recombinant APE1. Data were analyzed by non-linear regression. ( C ) CXCL1 G4-forming single-stranded oligo (CXCL1-G4), complementary C-rich single-stranded DNA oligo (CXCL1-G4 complement C-rich SSD), CXCL1 G4 double-stranded DNA oligo (CXCL1-G4 DSD), or Non-G4-random labeled with 50 nM biotin were incubated with increasing concentrations of recombinant APE1 in the presence of 50 mM KCl, and ELISAs were performed using APE1 antibody. Non-linear regression was used to analyze the data. ( D ) Representative images of tghe PLA show APE1 and G4 proximity (distance ≤ 40nm) in the nucleus in WT and APE1-KO TNBC cells. G4–APE1 PLA foci were visualized by confocal microscopy imaging (magnification: ×63). No antibody, anti-APE1 alone, G4 antibody alone, or BCL2 antibody, served as negative controls.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies: anti-APE1 (Novus, Cat# NB100-101), anti-Ki67 (Abcam, Cat# ab15580), anti-mouse IgG (Santa Cruz, Cat# sc-2025), and anti-rabbit IgG (Cell Signaling Technology, Cat# 2729).

Techniques: In Vitro, Labeling, Incubation, Recombinant, Confocal Microscopy, Imaging