Chris P. Ponting

Science Forum: The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Single-Cell Multiomics: Multiple Measurements from Single Cells

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cells phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

Identification of genetic variants affecting vitamin D receptor binding and associations with autoimmune disease

Abstract Large numbers of statistically significant associations between sentinel SNPs and case-control status have been replicated by genome-wide association studies. Nevertheless, few underlying molecular mechanisms of complex disease are currently known. We investigated whether variation in binding of a transcription factor, the vitamin D receptor (VDR), whose activating ligand vitamin D has been proposed as a modifiable factor in multiple disorders, could explain any of these associations. VDR modifies gene expression by binding DNA as a heterodimer with the Retinoid X receptor (RXR). We identified 43,332 genetic variants significantly associated with altered VDR binding affinity (VDR-BVs) using a high-resolution (ChIP-exo) genome-wide analysis of 27 HapMap lymphoblastoid cell lines. VDR-BVs are enriched in consensus RXR::VDR binding motifs, yet most fell outside of these motifs, implying that genetic variation often affects the binding affinity only indirectly. Finally, we compared 341 VDR-BVs replicating by position in multiple individuals against background sets of variants lying within VDR-binding regions that had been matched in allele frequency and were independent with respect to linkage disequilibrium. In this stringent test, these replicated VDR-BVs were significantly (q < 0.1) and substantially (>2-fold) enriched in genomic intervals associated with autoimmune and other diseases, including inflammatory bowel disease, Crohn’s disease and rheumatoid arthritis. The approach’s validity is underscored by RXR::VDR motif sequence being predictive of binding strength and being evolutionarily constrained. Our findings are consistent with altered RXR::VDR binding contributing to immunity-related diseases. Replicated VDR-BVs associated with these disorders could represent causal disease risk alleles whose effect may be modifiable by vitamin D levels.

The role of ADP-ribosylation in regulating DNA interstrand crosslink repair

ABSTRACT ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2. Summary: Here, we identify a role for post-translational modification ADP-ribosylation in the response to DNA interstrand crosslinks in the model Dictyostelium.

Biological function in the twilight zone of sequence conservation

Strong DNA conservation among divergent species is an indicator of enduring functionality. With weaker sequence conservation we enter a vast ‘twilight zone’ in which sequence subject to transient or lower constraint cannot be distinguished easily from neutrally evolving, non-functional sequence. Twilight zone functional sequence is illuminated instead by principles of selective constraint and positive selection using genomic data acquired from within a species’ population. Application of these principles reveals that despite being biochemically active, most twilight zone sequence is not functional.

Complexities of post-transcriptional regulation and the modeling of ceRNA crosstalk

ABSTRACT Control of gene and protein expression is required for cellular homeostasis and is disrupted in disease. Following transcription, mRNA turnover and translation is modulated, most notably by microRNAs (miRNAs). This modulation is controlled by transcriptional and post-transcriptional events that alter the availability of miRNAs for target binding. Recent studies have proposed that some transcripts – termed competitive endogenous RNAs (ceRNAs) – sequester a miRNA and diminish its repressive effects on other transcripts. Such ceRNAs thus mutually alter each other’s abundance by competing for binding to a common set of miRNAs. Some question the relevance of ceRNA crosstalk, arguing that an individual transcript, when its abundance lies within a physiological range of gene expression, will fail to compete for miRNA binding due to the high abundance of other miRNA binding sites across the transcriptome. Despite this, some experimental evidence is consistent with the ceRNA hypothesis. In this review, we draw upon existing data to highlight mechanistic and theoretical aspects of ceRNA crosstalk. Our intent is to propose how understanding of ceRNA crosstalk mechanisms can be improved and what evidence is required to demonstrate a ceRNA mechanism. A greater understanding of factors affecting ceRNA crosstalk should shed light on its relevance in physiological states. Graphical Abstract

The phenotypic spectrum of Xia-Gibbs syndrome

Xia‐Gibbs syndrome (XGS: OMIM # 615829) results from de novo truncating mutations within the AT‐Hook DNA Binding Motif Containing 1 gene (AHDC1). To further define the phenotypic and molecular spectrum of this disorder, we established an XGS Registry and recruited patients from a worldwide pool of approximately 60 probands. Additional de novo truncating mutations were observed among 25 individuals, extending both the known number of mutation sites and the range of positions within the coding region that were sensitive to alteration. Detailed phenotypic examination of 20 of these patients via clinical records review and data collection from additional surveys showed a wider age range than previously described. Data from developmental milestones showed evidence for delayed speech and that males were more severely affected. Neuroimaging from six available patients showed an associated thinning of the corpus callosum and posterior fossa cysts. An increased risk of both scoliosis and seizures relative to the population burden was also observed. Data from a modified autism screening tool revealed that XGS shares significant overlap with autism spectrum disorders. These details of the phenotypic heterogeneity of XGS implicate specific genotype/phenotype correlations and suggest potential clinical management guidelines.

ELIXIR-UK role in bioinformatics training at the national level and across ELIXIR

ELIXIR-UK is the UK node of ELIXIR, the European infrastructure for life science data. Since its foundation in 2014, ELIXIR-UK has played a leading role in training both within the UK and in the ELIXIR Training Platform, which coordinates and delivers training across all ELIXIR members. ELIXIR-UK contributes to the Training Platform’s coordination and supports the development of training to address key skill gaps amongst UK scientists. As part of this work it acts as a conduit for nationally-important bioinformatics training resources to promote their activities to the ELIXIR community. ELIXIR-UK also leads ELIXIR’s flagship Training Portal, TeSS, which collects information about a diverse range of training and makes it easily accessible to the community. ELIXIR-UK also works with others to provide key digital skills training, partnering with the Software Sustainability Institute to provide Software Carpentry training to the ELIXIR community and to establish the Data Carpentry initiative, and taking a lead role amongst national stakeholders to deliver the StaTS project – a coordinated effort to drive engagement with training in statistics.

Big knowledge from big data in functional genomics

With so much genomics data being produced, it might be wise to pause and consider what purpose this data can or should serve. Some improve annotations, others predict molecular interactions, but few add directly to existing knowledge. This is because sequence annotations do not always implicate function, and molecular interactions are often irrelevant to a cells or organisms survival or propagation. Merely correlative relationships found in big data fail to provide answers to the Why questions of human biology. Instead, those answers are expected from methods that causally link DNA changes to downstream effects without being confounded by reverse causation. These approaches require the controlled measurement of the consequences of DNA variants, for example, either those introduced in single cells using CRISPR/Cas9 genome editing or that are already present across the human population. Inferred causal relationships between genetic variation and cellular phenotypes or disease show promise to rapidly grow and underpin our knowledge base.