In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide ...transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery—persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
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•Specific RPB1 K1268 ubiquitylation targets RNAPII for UV-induced proteolysis•RPB1 K1268 ubiquitylation is required for surviving DNA damage•Control of the RNAPII pool via degradation regulates the transcriptome after UV•Lack of transcription recovery in Cockayne syndrome is caused by unstable RNAPII
Control of the pool of available RNA polymerase II shapes how cells respond to UV stress and the efficacy of the resulting damage response.
Lesions in genes that result in RNA polymerase II (RNAPII) stalling or arrest are particularly toxic as they are a focal point of genome instability and potently block further transcription of the ...affected gene. Thus, cells have evolved the transcription-coupled nucleotide excision repair (TC-NER) pathway to identify damage-stalled RNAPIIs, so that the lesion can be rapidly repaired and transcription can continue. However, despite the identification of several factors required for TC-NER, how RNAPII is remodelled, modified, removed, or whether this is even necessary for repair remains enigmatic, and theories are intensely contested. Recent studies have further detailed the cellular response to UV-induced ubiquitylation and degradation of RNAPII and its consequences for transcription and repair. These advances make it pertinent to revisit the TC-NER process in general and with specific discussion of the fate of RNAPII stalled at DNA lesions.
Health-care workers (HCWs) are at high risk of developing COVID-19, and may themselves contribute to transmission.1 To evaluate these risks, we enrolled 200 patient-facing HCWs between March 26 and ...April 8, 2020, in SARS-CoV-2 Acquisition in Frontline Healthcare Workers—Evaluation to inform Response (SAFER), a prospective cohort study in high-risk frontline HCWs in an acute National Health Service hospital trust in London. Of the 42 HCWs that ever tested positive for SARS-CoV-2 by RT-PCR, 20 (48%) reported symptoms within 7 days of the positive test that were consistent with Public Health England's COVID-19 case definition,2 and 16 (38%) did not report any symptoms in the same time frame. CS is funded by a Royal Society Napier Professorship, the Rosetrees Trust, and the Breast Cancer Research Foundation; receives grant support from Pfizer, AstraZeneca, BMS, Roche-Ventana, Boehringer Ingelheim, and Ono Pharmaceutical; has consulted for Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi, and the Sarah Cannon Research Institute; is a shareholder of Apogen Biotechnologies, Epic Bioscience, and GRAIL; and has stock options in and is co-founder of Achilles Therapeutics.
Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription
. This response is ...absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins
. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5
) and CSB (Csb
; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5
Csb
mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.
A variety of DNA repair pathways operate in different cellular contexts to tackle a diversity of DNA lesions and maintain genome stability. Nucleotide excision repair (NER) recognises and removes a ...variety of helix-distorting lesions, operating via two pathways: a global-genome (GG-NER) and transcription-coupled (TC-NER) pathway. GG-NER repairs damage at any locus in the genome thus promoting genome stability by abrogating replication-mediated stress and mutagenesis. TCNER on the other hand is restricted to the template strand of actively transcribed genes and provides means to rapidly repair lesions that would otherwise impair transcription. Thus, TC-NER has seemingly evolved to not only maintain genome stability but sustain transcription by removal of RNA polymerase II (RNAPII)-stalling lesions. The two pathways differ in their mode of recognition of lesions while downstream repair steps of excision and DNA synthesis are mutual. GG-NER relies on XPC and UV-DDB that recognise and bind directly to lesions, initiating repair. TC-NER is initiated by a lesion-stalled RNAPII, which is recognised by Cockayne's syndrome B (CSB) prompting recruitment of further repair factors. Mutations in CSB or CSA result in Cockayne's syndrome, a disease characterised by photosensitivity, neurological deficiencies and progeria. UV-DDB and CSA reside in ubiquitin ligase complexes highlighting the importance of ubiquitylation in NER. The Svejstrup laboratory previously identified a ubiquitinbinding domain in CSB that was essential for its function as well as several CSAand UV-dependent ubiquitylation sites on CSB. Building on this work, I have developed a cell system exclusively expressing CSB mutants that are not ubiquitylated in response to UV-irradiation. I present evidence that ubiquitylation of CSB is necessary for the recovery of transcription and cell survival following UVirradiation. Inhibition of CSB ubiquitylation does not affect its recruitment to chromatin following UV, indicating it is a step downstream of the recognition and binding of a stalled RNAPII. These data support the hypothesis that CSB ubiquitylation is a vital step in TC-NER.
Routine asymptomatic testing using RT-PCR of people who interact with vulnerable populations, such as medical staff in hospitals or care workers in care homes, has been employed to help prevent ...outbreaks among vulnerable populations. Although the peak sensitivity of RT-PCR can be high, the probability of detecting an infection will vary throughout the course of an infection. The effectiveness of routine asymptomatic testing will therefore depend on testing frequency and how PCR detection varies over time.
We fitted a Bayesian statistical model to a dataset of twice weekly PCR tests of UK healthcare workers performed by self-administered nasopharyngeal swab, regardless of symptoms. We jointly estimated times of infection and the probability of a positive PCR test over time following infection; we then compared asymptomatic testing strategies by calculating the probability that a symptomatic infection is detected before symptom onset and the probability that an asymptomatic infection is detected within 7 days of infection.
We estimated that the probability that the PCR test detected infection peaked at 77% (54-88%) 4 days after infection, decreasing to 50% (38-65%) by 10 days after infection. Our results suggest a substantially higher probability of detecting infections 1-3 days after infection than previously published estimates. We estimated that testing every other day would detect 57% (33-76%) of symptomatic cases prior to onset and 94% (75-99%) of asymptomatic cases within 7 days if test results were returned within a day.
Our results suggest that routine asymptomatic testing can enable detection of a high proportion of infected individuals early in their infection, provided that the testing is frequent and the time from testing to notification of results is sufficiently fast.
•We found a high asymptomatic rate in vulnerable people with epilepsy.•Enhanced surveillance allows to quickly contain outbreaks.•We report a low rate of COVID-19 morbidity and mortality in a ...long-term care facility.•Preventative measures allow reducing resident-to-resident and -to-caregiver transmission.•Children and young adults appear to have lower infection rates.
In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance.
During 25-week observation period (16 March–6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate.
We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth’s (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive.
Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes.
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