131 research outputs found
DNA lesion bypass and the stochastic dynamics of transcription coupled repair
DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage
CTCF maintains regulatory homeostasis of cancer pathways
Abstract
Background
CTCF binding to DNA helps partition the mammalian genome into discrete structural and regulatory domains. Complete removal of CTCF from mammalian cells causes catastrophic genome dysregulation, likely due to widespread collapse of 3D chromatin looping and alterations to inter- and intra-TAD interactions within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction of CTCF expression are viable, albeit with increased cancer incidence. Here, we exploit chronic Ctcf hemizygosity to reveal its homeostatic roles in maintaining genome function and integrity.
Results
We find that Ctcf hemizygous cells show modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these are enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, we observe dysregulation of the expression of several hundred genes, which are concentrated in cancer-related pathways, and are caused by changes in transcriptional regulation. Chromatin structure is preserved but some loop interactions are destabilized; these are often found around differentially expressed genes and their enhancers. Importantly, the transcriptional alterations identified in vitro are recapitulated in mouse tumors and also in human cancers.
Conclusions
This multi-dimensional genomic and epigenomic profiling of a Ctcf hemizygous mouse model system shows that chronic depletion of CTCF dysregulates steady-state gene expression by subtly altering transcriptional regulation, changes which can also be observed in primary tumors
Single-mitosis dissection of acute and chronic DNA mutagenesis and repair
How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes
Sex, Endothelial Cell Functions, and Peripheral Artery Disease
Peripheral artery disease (PAD) is caused by blocked arteries due to atherosclerosis and/or thrombosis which reduce blood flow to the lower limbs. It results in major morbidity, including ischemic limb, claudication, and amputation, with patients also suffering a heightened risk of heart attack, stroke, and death. Recent studies suggest women have a higher prevalence of PAD than men, and with worse outcomes after intervention. In addition to a potential unconscious bias faced by women with PAD in the health system, with underdiagnosis, and lower rates of guideline-based therapy, fundamental biological differences between men and women may be important. In this review, we highlight sexual dimorphisms in endothelial cell functions and how they may impact PAD pathophysiology in women. Understanding sex-specific mechanisms in PAD is essential for the development of new therapies and personalized care for patients with PAD
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Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry.
BACKGROUND: Optoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures. METHODS: Multispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO2MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation. RESULTS: MCF-7 tumours show SO2MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO2MSOT with a significant rim-core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry. CONCLUSIONS: OT is sensitive to differences in the vascular phenotypes of our breast cancer models
Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.This work was supported by a Wellcome Trust and Cancer Research UK (CRUK) Grand Challenge Award (C98/A24032). P.J.C. is a Wellcome Trust Senior Clinical Fellow (WT088340MA); S.F.B. was supported by the Swiss National Science Foundation (P2SKP3-171753 and P400PB-180790); M.A.S. is supported by a Rubicon fellowship from NWO (019.153LW.038); the Cambridge Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre; and M.H. is supported by a CRUK Clinician Scientist Fellowship (C52489/A19924)
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Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains
Funder: European Molecular Biology Laboratory; doi: http://dx.doi.org/10.13039/100013060Abstract: Background: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. Results: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. Conclusions: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure
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Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains
Funder: European Molecular Biology Laboratory; doi: http://dx.doi.org/10.13039/100013060Abstract: Background: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. Results: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. Conclusions: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure
Protocol for the stimulating β3-Adrenergic receptors for peripheral artery disease (STAR-PAD) trial: a double-blinded, randomised, placebo-controlled study evaluating the effects of mirabegron on functional performance in patients with peripheral arterial disease
Introduction: There is currently only one approved medication effective at improving walking distance in people with intermittent claudication. Preclinical data suggest that the β3-adrenergic receptor agonist (mirabegron) could be repurposed to treat intermittent claudication associated with peripheral artery disease. The aim of the Stimulating β3-Adrenergic Receptors for Peripheral Artery Disease (STAR-PAD) trial is to test whether mirabegron improves walking distance in people with intermittent claudication.
Methods and analysis: The STAR-PAD trial is a Phase II, multicentre, double-blind, randomised, placebo-controlled trial of mirabegron versus placebo on walking distance in patients with PAD. A total of 120 patients aged ≥40 years with stable PAD and intermittent claudication will be randomly assigned (1:1 ratio) to receive either mirabegron (50 mg orally once a day) or matched placebo, for 12 weeks. The primary endpoint is change in peak walking distance as assessed by a graded treadmill test. Secondary endpoints will include: (i) initial claudication distance; (ii) average daily step count and total step count and (iii) functional status and quality of life assessment. Mechanistic substudies will examine potential effects of mirabegron on vascular function, including brachial artery flow-mediate dilatation; MRI assessment of lower limb blood flow, tissue perfusion and arterial stiffness and numbers and angiogenesis potential of endothelial progenitor cells. Given that mirabegron is safe and clinically available for alternative purposes, a positive study is positioned to immediately impact patient care.
Ethics and dissemination: The STAR-PAD trial is approved by the Northern Sydney Local Health District Human Research Ethics Committee (HREC/18/HAWKE/50). The study results will be published in peer-reviewed medical or scientific journals and presented at scientific meetings, regardless of the study outcomes
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