Computational Analysis of Transcriptional Regulation

It is doubtful Friedrich Miescher appreciated how groundbreaking and transformative his isolation of ’nuclein’ in 1869 would prove. Eukaryotic gene expression is a noisy process that is subject to multiple layers of regulation. Key features of this are the three-dimensional (3D) chromatin organisation of eukaryotic genomes and the post-transcriptional control of RNA fates. Eukaryotic nuclear DNA is tightly packaged as chromatin that is further folded into higher order structures. The 3D folding of eukaryotic genome lends itself to the formation of interactions between otherwise distant regions of the genome. These interactions modulate transcription. I investigated the impact of human papillomavirus (HPV) 16 integration on host chromatin organisation and transcription using the W12 model for early cervical carcinogenesis with a novel Chromosome Conformation Capture (3C) method that specifically enriches for interactions involving viral integrants. Integration occurs without disrupting host 3D chromatin structure but alters the expression of many neighbouring host genes. The advent of reliable protocols for performing single-cell RNA sequencing (scRNA-seq) has revealed that transcriptional noise is widespread and a biologically important feature in many populations of mammalian cells. Ageing is associated with the progressive decline in biological function. It has recently been described that aged somatic tissues have greater cell-to-cell transcriptional variability. Ageing is also associated with a decline in male fertility. Some have attributed this to the clonal expansion of selfish spermatogonial lineages. To address this, I explored the effect of ageing on the transcriptomes of sorted populations of mouse undifferentiated spermatogonia using bulk and single-cell RNA sequencing (RNAseq). While subtle changes in mean gene expression are detectable, it was apparent that ageing, unlike in somatic tissues, leads to a decline in cell-to-cell transcriptional variability. This may reflect the phenomenon of selfish spermatogonial selection. Finally, I explored the role of an RNA post-transcriptional modification (RPTM), N6- methyladenosine (m6A), in buffering transcriptional noise. Maternally-supplied YTHDF2 is essential for degradation of m6A-modified transcripts during the maternal-to-zygotic transition (MZT) early in mammalian embryogenesis. YTHDF2 targets increase in abundance in its absence. Using scRNA-seq data generated from control and maternal conditional knock-out mouse zygotes I show that many of these targets exhibit greater cell-to-cell transcriptional heterogeneity in the absence of YTHDF2-mediated degradation. Suggesting that YTHDF2 has a additional function in buffering transcriptional noise.EMBL International PhD Programm

Lattice QCD and Particle Physics

Contribution from the USQCD Collaboration to the Proceedings of the US Community Study on the Future of Particle Physics (Snowmass 2021).Comment: 27 pp. main text, 4 pp. appendices, 30 pp. reference

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Lattice QCD and Particle Physics

Contribution from the USQCD Collaboration to the Proceedings of the US Community Study on the Future of Particle Physics (Snowmass 2021)

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

IL10RA modulates crizotinib sensitivity in NPM1-ALK-positive anaplastic large cell lymphoma

Anaplastic Large Cell Lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive Anaplastic Lymphoma Kinase (ALK) fusion protein. ALK inhibitors such as crizotinib provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by NPM1-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54-90% in clinical trials. However, a subset of patients progress within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide CRISPR activation and knockout screens in ALCL cell lines combined with RNA-seq data derived from ALK inhibitor relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of IL10RA. Elevated IL10RA expression rewires the STAT3 signaling pathway bypassing otherwise critical phosphorylation by NPM1-ALK. IL10RA expression does not correlate with response to standard chemotherapy in pediatric patients suggesting that combination of crizotinib with chemotherapy could prevent ALK-inhibitor resistance-specific relapse

Simultaneous sequencing of genetic and epigenetic bases in DNA.

Acknowledgements: We would like to acknowledge the practical assistance and helpful comments of current and former employees of Cambridge Epigenetix.DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine