Biology subjects

Bunch, H.

Publications and source records attributed to Bunch, H..

8 recordsLinked to original sources

bioRxiv2026

A functional triangle relationship of topoisomerase IIβ, BARD1, and ERK2 in immediate early gene transcription

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 II{beta} (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.

molecular biology↗

bioRxiv2025

Novel DNA endonuclease activity of human ERK1 protein

ERK1 is a key kinase in the mitogen-activated protein kinase pathway, which transduces extracellular signals into cellular responses. Following triggering of the pathway, cytosolic ERK1 is phosphorylated, translocates into the nucleus, and activates DNA-bound transcription factors and target genes. This study reports a novel, non-canonical role of human ERK1 as a DNA endonuclease. It catalyzes single-strand DNA breaks in sequence-unspecific yet, topology-sensitive manners. ERK1 catalysis requires magnesium ions and no ATP consumption. Comprehensive biochemical and in silico structural analyses indicate that a Mg2+ pocket, including a residue N171, which is allosterically modulated by the N-terminal domain, is important for DNA substrate binding and catalysis. Fluorescence imaging analysis suggests that ERK1 may interact with cytoplasmic DNAs in molecular condensates and destabilize them. Collectively, this study reveals an unprecedented role of ERK1 in nicking DNA molecules, which highlights the potential to regulate the fate of cytoplasmic DNAs.

molecular biology↗

bioRxiv2025

DNA topological regulation by topoisomerase IIbeta-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

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 II{beta} (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 HIF1 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.

molecular biology↗

bioRxiv2024

Jerusalem artichoke extracts regulate the gene expression of key enzymes involved in fatty acid biosynthesis

Jerusalem artichoke (JA) is a traditional remedy for alleviating symptoms of diabetes. In fact, the suppressive effects of JA on blood sugar have been reported in multiple studies since 1934. Recent studies have indicated that type II diabetes is often caused by insulin resistance rather than insulin reduction and that increased blood and interstitial fatty acid levels contribute to insulin resistance and the development of diabetes. However, whether JA affects lipogenesis has not been studied. Here, we elucidated the effects of JA on the expression of two key enzymes involved in fatty acid biosynthesis, fatty acid synthase (FASN) and acetyl-coA carboxylase (ACACA), using three immortalized human bone marrow, colon, and liver cell lines. Caffeine and ICRF193, a catalytic inhibitor of topoisomerase II (TOP2), were included as positive controls, and JA was extracted into water- or dimethyl sulfoxide-soluble components, termed H-JA and D-JA. D-JA significantly reduced the expression of FASN and ACACA at the mRNA and protein levels. D-JA-treated cells exhibited altered TOP2 levels and FASN/ACACA expression appeared to be controlled by TOP2 activity and levels. Taken together, our study revealed a novel effect of JA extracts on inhibiting the expression of the key enzymes involved in the fatty acid synthesis and suggested the potential of JA as a natural medicinal agent to control lipogenesis in humans. HighlightsO_LIJerusalem artichoke extracts reduce the expression of FASN and ACACA genes at the mRNA and protein levels C_LIO_LITopoisomerase II regulates FASN and ACACA gene expression C_LIO_LIJerusalem artichoke extracts and plasma glucose concentrations regulate cellular topoisomerase II protein expression C_LI

cell biology↗

bioRxiv2023

Lactiplantibacillus plantarum K8 lysates regulate hypoxia-induced gene expression

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1 accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1 target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. In addition, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1 destabilization under normoxic conditions. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1-mediated hypoxic gene activation.

molecular biology↗

bioRxiv2022

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

The transcription of stress-inducible genes requires synchronized and robust activation, which is critical for organismal survival and homeostasis. The function of the mitogen-activated protein kinase (MAPK) signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation1-3. In addition, recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs4-6. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK1 can relax the DNA by itself and produces a semi-relaxed DNA, which is apparently resistant to TOP2B catalysis. Inhibition of ERK2 kinase activity or ERK2 knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we obtained the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (50 nt; -30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure elucidated in our studies showed TOP2B-mediated breakage and dramatic bending of the double-stranded DNA, comparable to previously reported structures of TOP2. Our cell-based analyses showed transcriptional activation by etoposide and transcriptional inhibition by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

molecular biology↗

bioRxiv2021

Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans [1, 2]. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models [3, 4]. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

genetics↗

bioRxiv2020

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase II{beta} (TOP2B)-mediated DNA strand break and the activation of DNA damage response signaling 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 ATM and ATR during gene activation and that this event is essential for productive transcription. Our in vitro biochemical analyses showed TOP2B and BARD1 interaction and colocalization in the EGR1 transcription start site (TSS) and that the BRCA1-BARD1 complex ubiquitinates TOP2B, which appears to stabilize TOP2B protein in the cell and binding to DNA. Intriguingly, BRCA1 phosphorylation at S1524 controls this interaction. In addition, genomic analyses indicated colocalization between TOP2B and BRCA1 in a large number of protein-coding genes. Together, these findings reveal the novel function of the BRCA1-BARD1 complex in gene expression and in the regulation of TOP2B during Pol II transcription. Significance StatementMaintaining genomic integrity against cellular and extracellular genotoxic elements is essential for normal cell growth and function. Recent studies indicated that stimulus-induced transcription provokes topoisomerase II{beta}-mediated DNA strand break and DNA damage response signaling, requiring DNA repair to be coupled with transcription. Here, we present a novel role for the BRCA1-BARD1 complex in regulating the transcription of serum-inducible genes and the stability of topoisomerase II{beta}. The mechanism involving topoisomerase II{beta} ubiquitination by the BRCA1-BARD1 complex and the phosphorylation of BRCA1 S1524 upon transcriptional activation appears to function as a switch to the reaction. Our findings provide the first evidence of functional interaction between the BRCA1-BARD1 complex and topoisomerase II{beta} in transcription in humans.

molecular biology↗