Fig 2: 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.

Fig 3: BRCA1 phosphorylation controls TOP2B ubiquitination and DNA-binding affinity. (a) Purified TOP2Bubi (ubi) and TOP2Bdeubi (Deu) shown by silver staining and immunoblotting. (b) EMSA comparing TOP2Bubi versus TOP2Bdeubi 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 KD 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.

Fig 4: 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.

Fig 5: 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 G0; 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 a-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. ß-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.