BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

Abstract

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

“…1B ). In addition, it has been previously shown that TOP2B is recruited to IEGs, including the EGR1 gene, upon transcriptional activation 5,6 . Moreover, TOP2B inhibition using ICRF193 suppresses Pol II pause release and productive transcription in these genes 5 .…”

“…The EGR1 template DNA, –423 to +332, was cloned into a pCR-Blunt-TOPO to generate pTOPO-EGR1 6 . The biotinylated EGR1 template was PCR-amplified using a pair of primers, one conjugated with biotin at the 5’ end (Table S1), and prepared using the same method described in our previous study 6 . ELK1m was constructed using a set of primers (Table S1), introducing the desired mutation, and pTOPO-EGR1 as a template.…”

“…The cells were lysed with the xTractor Bacterial Cell Lysis Buffer (Clontech) and sonicated. His 6 -tagged ELK1 was purified on a Ni affinity column using the same method as previously described 6,24 . The purified proteins were validated by SDS-PAGE and immunoblotting (see the antibody information below).…”

“…For TOP2B inhibitors, ICRF193 (Sigma, I4659) and etoposide (Sigma, 341205) were used at a final concentration of 10 µM in 0.1% DMSO for 3 h. An ERK2 inhibitor, VX-11e (Selleck, S7709) was used at a final concentration of 100 nM in 0.1% DMSO for 3-5 h. Control cells were treated with the same amount of DMSO for the same duration. Serum starvation and induction were performed as previously described 5,6 .…”

“…Human full-length TOP2B was purified from HEK cells, as previously described 6,66 . For the in vitro kinase assay, mass spectrometry, and cryo-EM, ubiquitin-stripped TOP2B 6 was used. The activity of purified TOP2B was confirmed using an in vitro decatenation assay, as previously described 6 .…”