Protein synthesis is a multi-stage process. The transfer of the genetic instructions stored in DNA and needed to make polypeptides to messenger RNA (mRNA) molecules is called transcription. Transcription simply converts the genetic information to these intermediary molecules that are used for the synthesis of proteins by a complex process called translation. Overall, the production of a functional protein encoded in a gene is referred to as gene expression. Gene expression can be modulated at any stage to ensure the maintenance of the cellular processes, including developmental changes, proliferation, and cellular responses to external stimuli.
Accurate transcription is essential for preserving genome integrity and proper gene expression. The cells evolve complex pathways collectively establishing DNA damage response (DDR) signalling to respond to any genotoxic mechanisms that compromise the genomic fidelity. While various repair protein complexes are activated in different types of DNA lesions – typically the kinases including ataxia telangiectasia mutated (ATM) – ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) are activated upon the detection of DNA double strand break. These kinases phosphorylate their targets at the sites of the DNA break.
Breast cancer 1 (BRCA1) is an effector for precise DNA double strand break repair. The kinases ATM and ATR mediate the phosphorylation of BRCA1 at several residues in response to DNA damage. BRCA1 partners with BRCA1-associated ring domain 1 (BARD1) to form the BRCA1-BARD1 heterodimer with an ability to conjugate ubiquitin to protein substrates. This E3 ubiquitin ligase activity can link the protein to altered activities or destruction pathways. Though repair mechanisms are activated upon generation of DNA lesions, DNA breaks might be unrepairable, the genome is mutated, and the cell undergoes programmed cell death if the damage is too severe.
RNA polymerase II promoter-proximal pausing for prompt and decisive cell regulation
In eukaryotes, there are three RNA polymerases catalysing the transcription from DNA to RNA. Among them, RNA polymerase II (Pol II) targets all protein-coding genes and many noncoding RNAs. Transcription initiation requires the association of DNA-binding transcription factors (TFs) with their target motifs and the employment of Pol II and general TFs to position Pol II correctly at the promoter region, where transcription begins. For years, initiation, elongation along the full length of the gene and termination were considered the major steps of transcription that are tightly controlled by various protein and nucleic acid factors. Intriguingly, gene expression can also be regulated in early elongation prior to processive elongation, where Pol II pauses at around nucleotides +25–100 relative to the transcription start site (TSS), awaiting further signals to induce elongation. Only, the controlled release of paused Pol II produces a functional full-length mRNA. This phenomenon, namely Pol II promoter-proximal pausing, represents a major checkpoint for proper gene expression and is exhibited by approximately over 30% of coding genes and more than 70% of developmental or stimulus-inducible genes.
A novel finding
Despite decades of research since its discovery in the late 1980s, the mechanisms underlying stable Pol II pausing and release aren’t fully understood. Back in 2014, Dr Heeyoun Bunch and her colleagues at the Beth Israel Deaconess Medical Center and Harvard Medical School identified TRIM28, a TF and DDR protein, as a modulator of Pol II pausing and transcriptional elongation. The researchers conducted the initial studies on the human heat shock protein 70 (HSP70) gene and demonstrated that the above-mentioned processes involved the phosphorylation of TRIM28 by the prominent DDR kinases ATM and DNA-PK. Moreover, by performing genome-wide analysis, they proposed a global function for TRIM28 in controlling Pol II pausing and pause release in mammalian genes.
The findings on TRIM28 prompted Bunch and colleagues to investigate a potential involvement of DNA breaks and DDR signalling in Pol II pause release and transcriptional activation. Their research in 2015 has shed new light on the topic and presented the coupling between DDR signalling and transcriptional elongation in stimulus-inducible protein coding genes as a novel mechanism for regulation of gene expression. Importantly, the researchers proposed that the enzyme topoisomerase II (TOP2B) mediates DNA double strand breaks and alters the topologic states of DNA for Pol II pause release and subsequent transcriptional elongation. TOP enzymes are responsible for the formation of transient physiological DNA breaks during normal cellular processes. They control DNA topology, and by binding to DNA and cutting the phosphate backbone, they also allow RNA polymerase action during transcription. Bunch and her colleagues raised great interest by presenting the involvement of TOP2B in transcriptional activation-coupled DNA breaks and repair signalling.
Repositioning of BRCA1-BARD1 complex
After establishing a link between DDR signalling and transcriptional elongation, the group further explored the role of DDR signalling in stress-inducible gene expression. To do that, they evaluated the function of the BRCA1-BARD1 complex in Pol II elongation of several human immediate early genes. These genes represent transcription factors and proto-oncogenes that are promptly activated in response to appropriate signals and stimuli such as cell proliferation signals. The transcription of these genes involves Pol II promoter-proximal pausing and stimulus-induced pause release.
The extensive experimentation, carefully designed to uncover the mechanisms of transcriptional regulation, indicated a new function for the BRCA1-BARD1 complex involved in DDR signalling through modulating the affinity of TOP2B to DNA. First, the researchers measured the ATM and ATR-induced phosphorylation of BRCA1 and demonstrated that BRCA1 is activated in response to DNA damage with phosphorylation during transcriptional elongation of human immediate early genes JUN, FOS, MYC and early growth response factor 1 (EGR1). BRCA1 in heterodimer form with BARD1 can bind near the TSS of these genes for modulating their transcription. Based on their findings, Bunch and her colleagues proposed a model involving the resting and active states of transcription: during the resting stage, Pol II is paused in the promoter-proximal site in human immediate early genes. The BRCA1-BARD1 complex physically interacts with TOP2B and drives the ubiquitination of the enzyme by functioning as an E3 ligase. This ubiquitination leads to a tight association of the highly stable enzyme with TSS. On the other hand, when ATM/ATR phosphorylates BRCA1 during activation of transcription, the opposite takes place: BRCA1 phosphorylation changes the aforementioned physical interaction and alters/reduces the TOP2B ubiquitination, which in turn will decrease the affinity of TOP2B to the TSS of the transcriptionally active gene. Overall, depending on its phosphorylation status, the DDR signalling complex BRCA1-BARD1 modulates transcription elongation by interacting with TOP2B.
In their latest work, Bunch and colleagues presented the BRCA1-BARD1 complex-mediated TOP2B regulation for modulation of stimulus-induced gene expression. They demonstrated not only the involvement of a DDR signalling molecules in early transcriptional regulation, but they also uncovered the mechanisms by which the BRCA1-BARD1 complex functions as an E3 ligase for altering the stability of TOP2B and its affinity to DNA during Pol II pause and pause release. Bunch’s work raises great attention by offering new insight into the early transcriptional regulation of stimulus-induced genes by assigning a novel role to the DDR signalling complex BRCA1-BARD1.
- Bunch, H, et al, (2021) BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ, Open Biology, 11(10), 210221. doi.org/10.1098/rsob.210221
- Molinaro, C, Martoriati, A, Cailliau, K, (2021) Proteins from the DNA damage response: regulation, dysfunction, and anticancer strategies. Cancers, 13(15), 3819.
- Noe Gonzalez, M, Blears, D, Svejstrup, JQ, (2021) Causes and consequences of RNA polymerase II stalling during transcript elongation. Nature Reviews. Molecular Cell Biology, 22(1), 3–21.
- Core, L, Adelman, K, (2019) Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes & Development, 33(15–16), 960–982.
- Densham, RM, Morris, JR, (2017) The BRCA1 ubiquitin ligase function sets a new trend for remodelling in DNA repair, Nucleus, 8(2), 116–125. doi.org/10.1080%2F19491034.2016.1267092
- Bunch, H, et al, (2015) Transcriptional elongation requires DNA break-induced signalling, Nature Communications, 6(1), 1–12. doi.org/10.1038/ncomms10191
- Bunch, H, et al, (2014) TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release, Nature Structural & Molecular Biology, 21(10), 876–883. doi.org/10.1038/nsmb.2878
Dr Bunch reported a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in transcriptional regulation.
National Research Foundation of the Republic of Korea
- Drs Stuart K Calderwood (Beth Israel Deaconess Medical Center, Harvard Medical School, USA)
- Matthew J Schellenberg (Mayo Clinic, USA)
- Benjamin P C Chen (University of Texas Southwestern Medical Center, USA)
Heeyoun Bunch acquired a PhD in microbiology at Cornell University (2009) and did her postdoctoral research at the University of Colorado (2010–2011), Harvard Medical School (2012–2016), and the University of Texas Southwestern Medical Center (2016–2017).
80 Daehak-ro, Kyungpook National University, Department of Applied Biosciences, Daegu 41566, Republic of Korea