Bunch, Heeyoun

ABSTRACT Immediate early genes (IEGs) encode master transcription factors, including EGR1 , FOS , JUN , and MYC genes that drive robust transcription in the G1 phase of cell cycle. Our previous studies indicate that topoisomerase IIβ (TOP2B) critically regulates IEG transcription and that the activities of TOP2B are dynamically controlled through post-translational modifications by BRCA1-BARD1 and ERKs. Here, we show that two E2 enzymes, UBCH5b and UBC13/MMS2, differentiate the effects and functions of TOP2B ubiquitination by BRCA1-BARD1. Comprehensive transcriptomics, proteomics, and biochemical and molecular cellular analyses revealed a close relationship between BARD1 and key mitogen-activated protein kinase pathway genes and identified activated ERK2 as a novel kinase that phosphorylates BARD1 at S391, a previously reported mitotic phosphorylation site, whose genetic mutation has been linked to tumorigenesis. Mechanistically, the catalytic activity of ERK2 stimulates TOP2B ubiquitination mediated by BRCA1-BARD1 in complex with UBCH5b and UBC13/MMS2, which controls the binding and function of TOP2B and BARD1 for transcriptional activation at representative IEGs. Taken together, our data propose that there is a functional regulatory circuit involving TOP2B, BARD1, and ERK2, three key transcriptional activators for IEG transcription, in which the gene association and catalytic activity of TOP2B are regulated through E2-differentiated ubiquitination by BRCA1-BARD1 and the phosphorylation of BARD1 by ERK2 for productive transcription.

ABSTRACT Hypoxic stress responses are essential for cellular and organismal survival and drive gene regulation across diverse biological pathways, including cell cycle progression and energy metabolism. Here, we show that topoisomerase IIβ (TOP2B) regulates DNA topology and transcription of hypoxia-inducible genes (HIGs) in a DNA-dependent protein kinase (DNA-PK)-dependent manner. Integrated cellular, biochemical, and genomic analyses reveal an antagonistic yet correlated relationship between TOP2B and DNA-PK activities. Under normoxic conditions, TOP2B associates with HIGs and represses transcription by suppressing DNA negative supercoiling formation and accessibility. Under hypoxic exposure, TOP2B dissociates from HIGs, while DNA-PK and HIF1α are recruited to activate HIGs. Notably, DNA-PK is required for the repressive function of TOP2B, as DNA-PK knockout abrogates TOP2B release and activity, resulting in elevated expression of a number of HIGs. Mechanistically, DNA-PK phosphorylates TOP2B at T1403, stimulating its catalytic activity to restrain DNA unwinding and accessibility, thereby suppressing HIG transcription. Collectively, these findings identify a novel role for TOP2B and DNA-PK-mediated regulation of TOP2B as key transcriptional regulators of HIG expression. We propose that phosphorylation-dependent modulation of TOP2B activity is coordinated with transcriptional states and determines DNA topology to regulate Pol II transcription.

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.

Recent studies indicated that activated transcription of serum‐inducible genes in humans requires topoisomerase IIβ (TOP2B)‐mediated DNA strand break (DSB) and DNA damage response (DDR) signaling. However, the mechanisms by which TOP2B is regulated in this process are unknown. Here, we identified a novel role of BRCA1‐BARD1 complex in transcription of these genes by regulating TOP2B binding to the transcription start site (TSS). Our genomic and in vitro biochemical analyses demonstrated that BRCA1‐BARD1 complex and TOP2B colocalize in a large number of genes and physically interact for TOP2B ubiquitination. Upon transcriptional activation, BRCA1 is phosphorylated at S1524 by ataxia telangiectasia‐mutated (ATM) and ataxia telangiectasia and Rad3‐related protein (ATR). Intriguingly, unphosphorylatable BRCA1 (S1524A)‐BARD1 complex ubiquitinates TOP2B, but not WT or S1524D BRCA1. Using the TSS of a representative serum‐inducible gene, EGR1 , we found that ubiquitinated TOP2B binds to the DNA with much more stability than deubiquitinated one does. Together, our findings suggest that BRCA1‐BARD1 complex stabilizes TOP2B association with serum‐inducible genes through its E3 ligase function in transcription. Transcriptional activation induces TOP2B‐mediated DSB and DDR signaling to activate ATM/ATR that phosphorylates BRCA1 S1524. Phosphorylated BRCA1 is ineffective in ubiquitinating TOP2B, which loosens it from the DNA. We propose that this loosening may contribute to either mobility or removal of TOP2B.