Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1-BARD1 ubiquitinates TOP2B in a phosphorylation-dependent manner. ( a ) (i) Immunoprecipitation with control IgG against 5 mg of HeLa NE. P, pellet (bound) and S, supernatant (unbound) fraction. Pellet and supernatant were loaded at 1 : 10 and 1 : 100 inputs, respectively. H chain, IgG heavy chain. (ii) Immunoprecipitation of TOP2B antibody against HeLa NE, followed by immunoblotting. ( b ) Immunoblotting data showing that BARD1 KD decreases the level of TOP2B proteins and ubiquitination. CDK9 was used as a loading control. HeLa NE was included as a technical control. ( c ) In vitro ubiquitination assay followed by immunoblotting showing discrete bands at about 56, 68, 81 and 91 KDa for 0, 1 (U•), 2 (U••) and 3 (•••) ubiquitin proteins ligated to TOP2B 1–566 by SA BRCA1-BARD1. W, WT BRCA1; A, SA BRCA1; D, SD BRCA1. ( d ) In vitro ubiquitination assay and immunoblotting screening the E2 enzymes, UBCH1, UBCH3, UBCH5b, UBCH6 and UBCH13/MMS2 for the ubiquitination of TOP2B deubi in collaboration with SA BRCA1-BARD1. Red boxes indicate UBCH5b and UBCH13/MMS2 to collaborate with the BRCA1-BARD1 complex to ubiquitinate TOP2B.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Ubiquitin Proteomics, In Vitro

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 regulates serum-induced transcriptional activation. ( a ) Schematic overview of cell cycle synchronization and serum-induced transcriptional activation in hIEGs with or without chemical kinase inhibitors. FBS, fetal bovine serum. ( b ) ChIP-qPCR showing BRCA1 (i) and pBRCA1 (ii) occupancy at FOS , MYC , JUN and EGR1 . S0, serum-starved cells at G 0 ; S15, serum-induced cells. Error bars show standard deviations (s.d., n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. ( c ) BRCA1 and BARD1 KD using siRNA species. (i), immunoblotting results showing the protein level of BRCA1, BARD1 and α-tubulin (tubulin, loading control, 70 µg cell lysate/lane) using siRNA species targeting BRCA1 (si-BR1) or BARD1 (si-BD1). SCR, scrambled siRNA. (ii, iii), BRCA1 and BARD1 mRNA expression in SCR control versus BRCA1 and BARD1 KD cells. Error bars show s.d. ( n = 3). ** p < 0.005. ( d ) qRT-PCR data showing the effects of BRCA1 or BARD1 KD on EGR1 (i), JUN (ii) and FOS (iii) mRNA expression. β-actin was used as a reference gene. Error bars show s.d. ( n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01. ( e ) ChIP-qPCR showing impaired Pol II, S2 Pol II and TOP2B recruitment upon gene activation at EGR1 (i), MYC (ii), FOS (iii) and JUN (iv) in BRCA1 KD cells. Error bars show s.d. ( n ≥ 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Activation Assay, ChIP-qPCR, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 is phosphorylated by ATM and ATR upon transcriptional activation. ( a ) ChIP-qPCR of pBRCA1 with or without KU55933 (ATMi) in the EGR1 TSS. Error bars show s.d. ( n = 2). * p < 0.05. ( b ) ChIP-qPCR of pBRCA1 with or without VE-821 (ATRi) in EGR1 TSS. Error bars show s.d. ( n = 2). * p < 0.05. ( c ) ChIP-qPCR showing that caffeine, which inhibits both ATM and ATR, alleviates the accumulation of pBRCA1 on EGR1 TSS upon transcriptional activation. Error bars show s.d. ( n = 3). * p < 0.05. ( d ) Immunoblotting showing that caffeine reduces the cellular level of pBRCA1 overall and upon transcriptional activation. α-tubulin was used as a loading control. ( e ) qRT-PCR showing the reduction of EGR1 mRNA level in caffeine-treated HEK293 cells upon transcriptional activation. β-actin was used as a reference and normalizer. Error bars show in s.d. ( n = 3). * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Activation Assay, ChIP-qPCR, Western Blot, Control, Quantitative RT-PCR

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 phosphorylation at S1524 is important for transcriptional activation. ( a ) Schematic overview of immobilized template and in vitro transcription assays. ( b ) Validation of PIC formation on the EGR1 TSS (–423 to +332) using immunoblotting. ( c ) In vitro transcription assay using recombinant WT, S1524A (SA), and S1524D (SD) BRCA1 showing that SD activates EGR1 transcription. ( d ) Quantification of the efficiency of S1524A and S1524D BRCA1 mutant proteins in stimulating transcription, relative to that of WT BRCA1. Error bars show s. d. ( n = 3). ** p < 0.002, * p < 0.02. ( e ) Immunofluorescence-confocal microscopy results showing increased levels of TOP2B and pBRCA1 in nuclei upon etoposide treatment. pBRCA1 intensity was quantified. n = 65. **** p < 0.0001. ( f ) Chromosome views of total BRCA1 (red) and TOP2B (etoposide-trapped, blue), and input control (grey) on representative hIEGs, EGR1 , JUN , MYC and FOS , illuminating genomic colocalization and functional collaboration. Black bars indicate binding sites of a given factor, identified by HOMER with a false discovery rate (FDR)-adjusted p -value of 0.001.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Activation Assay, In Vitro, Biomarker Discovery, Western Blot, Transcription Assay, Recombinant, Mutagenesis, Immunofluorescence, Confocal Microscopy, Control, Functional Assay, Binding Assay

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: TOP2B active (etoposide-captured) and BRCA1-binding sites are largely overlapped genome-wide. ( a ) Venn diagram showing the overlap of BRCA1 and TOP2B-binding sites in MCF10A cells. ( b ) Heat maps of the genes co-occupied by TOP2B and BRCA1 ( n = 6587).

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Binding Assay, Genome Wide

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: TOP2B and BARD1 bind to EGR1 TSS between –132 and –15. ( a ) DNA sequence of EGR1 TSS (–132 to +332). Orange flash signs indicate the restriction enzyme sites for NruI, SfoI and SacII used to map the factor binding region. Coloured letters indicate those that were subjected to mutations for the purposes indicated in the text. Altered sequences are presented under the original ones. TOP2B and BARD1 mutual binding site mapped in this study was boxed with light green. ( b ) Immobilized template assay combined with restriction enzyme digestion using EGR1 TSS (–423 to +332) and SacII (Sac, to digest at +92), followed by immunoblotting. SacII added immediately after PIC formation or NTP addition. Un, undigested template DNA; I.B., immunoblotting probing BARD1 and TOP2B; DNA, native PAGE detecting the released DNA fragment (241 nt) after SacII digestion; Silver, silver staining visualizing the proteins bound on the DNA. P, pellet; S, supernatant fraction. ( c ) Immobilized template assay using the EGR1 template (−132 to +332) in the presence or absence of BRCA1, combined with restriction enzyme digestion with NruI and SfoI that cut at −15 and +68, respectively. Recombinant WT BRCA1 was added at T2, immediately before the template was digested by restriction enzymes. The pellet and supernatant fractions were analysed by immunoblotting. ( d ) (i) Diagram of the four EGR1 TSS fragments. (ii) EMSA followed by silver staining showing TOP2B ubi binding to EGR1 TSS fragments with different affinities. The strongest binding was observed with EGR1 TSS no. 3 (–132 to +62). SM, size marker; P, TOP2B ubi . The second lane shows TOP2B ubi only.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Sequencing, Binding Assay, Western Blot, Clear Native PAGE, Silver Staining, Recombinant, Marker

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 phosphorylation controls TOP2B ubiquitination and DNA-binding affinity. ( a ) Purified TOP2B ubi (ubi) and TOP2B deubi (Deu) shown by silver staining and immunoblotting. ( b ) EMSA comparing TOP2B ubi versus TOP2B deubi for their binding affinity to EGR1 TSS no. 3 (–132 to +62). Silver-stained. SM, DNA size marker. ( c ) Ubiquitinated TOP2B binds to DNA with much higher affinity than deubiquitinated one. A plot summarizing EMSA to derive K D values. ( d ) Immobilized template assay results. Tight TOP2B association with EGR1 TSS (Pellet, –423 to +332) before NTP addition, regardless of recombinant BRCA1 species added at T1 during PIC formation. ( e ) Immobilized template assay results. Comparison of the degrees of TOP2B associated (Pellet, P) with and dissociated from EGR1 TSS (Supernatant, S) when WT, SA and SD BRCA1 were added after NTP addition (T2). ( f ) Quantification of TOP2B in pellet (DNA bound, i) and in supernatant (released, ii) in immobilized template assays. Error bars in s.d. ( n = 3). *** p < 0.005, ** p < 0.01, * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Binding Assay, Purification, Silver Staining, Western Blot, Staining, Marker, Recombinant, Comparison

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: Model of the BRCA1-BARD1 complex-mediated TOP2B regulation. During the resting state of transcription, Pol II is paused in the promoter-proximal site in hIEGs. The pausing is induced and stabilized by various factors including transcription factors, nucleosome modifiers and nucleic acids. The BRCA1-BARD1 complex that is engaged with hIEGs interacts with and ubiquitinates TOP2B in the EGR1 TSS. This ubiquitination (marked as ub) confers an enhanced DNA-binding affinity and stability to TOP2B (Tight TOP2B). In transcriptional activation, ATM/ATR phosphorylates its substrates including BRCA1 at S1524 (phosphorylation, marked as P in a blue circle). Phosphorylated BRCA1 alters the functional interaction between BRCA1-BARD1 and TOP2B to mitigate TOP2B ubiquitination. This event destabilizes and loosens TOP2B (Loose TOP2B) from the TSS. The interplay between BRCA1-BARD1 complex and TOP2B appears to be crucial in transcriptional regulation of hIEGs. Lightning marks are proposed catalysis sites of TOP2B.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Binding Assay, Activation Assay, Phospho-proteomics, Functional Assay