Improved simultaneous mapping of epigenetic features and 3D chromatin structure via ViCAR.

Methods to measure chromatin contacts at genomic regions bound by histone modifications or proteins are important tools to investigate chromatin organization. However, such methods do not capture the possible involvement of other epigenomic features such as G-quadruplex DNA secondary structures (G4s). To bridge this gap, we introduce ViCAR (viewpoint HiCAR), for the direct antibody-based capture of chromatin interactions at folded G4s. Through ViCAR, we showcase the first G4-3D interaction landscape. Using histone marks, we also demonstrate how ViCAR improves on earlier approaches yielding increased signal-to-noise. ViCAR is a practical and powerful tool to explore epigenetic marks and 3D genome interactomes.

Novel In-Line Hemolysis Detection on a Blood Gas Analyzer and Impact on Whole Blood Potassium Results.

BACKGROUND: Preanalytical error due to hemolyzed blood samples is a common challenge in laboratory and point-of-care (POC) settings. Whole blood potassium (K+) measurements routinely measured on blood gas analyzers are particularly susceptible to hemolysis, which poses a risk for incorrect K+ results. The GEM Premier 7000 with IQM3 (GEM 7000) blood gas analyzer provides novel integrated hemolysis detection within the sample measurement process. Therefore, the GEM 7000 can detect and flag hemolyzed whole blood samples at the POC, warning the operator of potentially erroneous results. METHODS: Heparinized venous or arterial whole blood samples were used for K+ interference studies and assessed for hemolysis agreement utilizing either a traditional volumetric method or chemistry analyzer serum index measurements with the Roche cobas c311 or Abbott Alinity c. RESULTS: Hemolysis interference studies performed at 2 different K+ concentrations (3.8 and 5.3 mmol/L) identified that a plasma free hemoglobin ≥116 mg/dL can impact K+ results on the GEM 7000. Hemolysis agreement studies demonstrated an excellent agreement of >99% with the volumetric method, 98.8% with cobas H index, and 96.4% with Alinity H index. GEM 7000 K+ results were correctly flagged for both native and spiked samples. CONCLUSION: GEM 7000 hemolysis detection provides a novel technology to detect hemolysis in whole blood samples. Moreover, the GEM 7000 demonstrates excellent agreement with traditional laboratory hemolysis detection methods and offers an integrated technological solution for assuring the quality of whole blood K+ results in POC settings.

Selective Photocatalytic C-H oxidation of 5-methylcytosine in DNA.

Selective C-H activation on complex biological macromolecules is a key goal in the field of organic chemistry. It requires thermodynamically challenging chemical transformations to be delivered in mild, aqueous conditions. 5-Methylcytosine (5mC) is a fundamentally important epigenetic modification in DNA that has major implications for biology and has emerged as a vital biomarker. Selective functionalisation of 5mC would enable new chemical approaches to tag, detect and map DNA methylation to enhance the study and exploitation of this epigenetic feature. We demonstrate the first example of direct and selective chemical oxidation of 5mC to 5-formylcytosine (5fC) in DNA, employing a photocatalytic system. This transformation was used to selectively tag 5mC. We also provide proof-of-concept for deploying this chemistry for single-base resolution sequencing of 5mC and genetic bases adenine (A), cytosine (C), guanine (G), thymine (T) in DNA on a next-generation sequencing system. This work exemplifies how photocatalysis has the potential to transform the analysis of DNA.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

An Upstream G-Quadruplex DNA Structure Can Stimulate Gene Transcription.

Four-stranded G-quadruplexes (G4s) are DNA secondary structures that can form in the human genome. G4 structures have been detected in gene promoters and are associated with transcriptionally active chromatin and the recruitment of transcription factors and chromatin remodelers. We adopted a controlled, synthetic biology approach to understand how G4s can influence transcription. We stably integrated G4-forming sequences into the promoter of a synthetic reporter gene and inserted these into the genome of human cells. The integrated G4 sequences were shown to fold into a G4 structure within a cellular genomic context. We demonstrate that G4 structure formation within a gene promoter stimulates transcription compared to the corresponding G4-negative control promoter in a way that is not dependent on primary sequence or inherent G-richness. Systematic variation in the stability of folded G4s showed that in this system, transcriptional levels increased with higher stability of the G4 structure. By creating and manipulating a chromosomally integrated synthetic promoter, we have shown that G4 structure formation in a defined gene promoter can cause gene transcription to increase, which aligns with earlier observational correlations reported in the literature linking G4s to active transcription.

G-quadruplex DNA structure is a positive regulator of MYC transcription.

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.

G-quadruplex is critical to epigenetic activation of the lncRNA HOTAIR in cancer cells.

The cancer-promoting lncRNA HOTAIR has multiple isoforms. Which isoform of HOTAIR accounts for its expression and functions in cancer is unknown. Unlike HOTAIR's canonical intergenic isoform NR_003716 (HOTAIR-C), the novel isoform NR_047517 (HOTAIR-N) forms an overlapping antisense transcription locus with HOXC11. We identified HOTAIR-N as the dominant isoform that regulates the gene expression programs and networks for cell proliferation, survival, and death in cancer cells. The CpG island in the HOTAIR-N promoter was marked with epigenetic markers for active transcription. We identified a G-quadruplex (G4) motif rich region in the HOTAIR-N CpG island. Our findings indicate that G4s in HOTAIR-N CpG island is critical for expression of HOTAIR-N in cancer cells. Disruption of G4 may represent a novel therapeutic approach for cancer. The transcriptomes regulated by HOTAIR-N and Bloom in cancer cells as provided herein are important resources for the exploration of lncRNA, DNA helicases, and G4 in cancer.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

A Photoredox Reaction for the Selective Modification of 5-Carboxycytosine in DNA.

Covalent epigenetic modifications contribute to the regulation of important cellular processes during development and differentiation, and changes in their genomic distribution and frequency are linked to the emergence of genetic disease states. Chemical and enzymatic methods that selectively target the orthogonal chemical functionality of epigenetic markers are central to the study of their distribution and function, and considerable research effort has been focused on the development of nondestructive sequencing approaches which preserve valuable DNA samples. Photoredox catalysis enables transformations with tunable chemoselectivity under mild, biocompatible reaction conditions. We report the reductive decarboxylation of 5-carboxycytosine via a novel iridium-based treatment, which represents the first application of visible-light photochemistry to epigenetic sequencing via direct base conversion. We propose that the reaction involves an oxidative quenching cycle beginning with single-electron reduction of the nucleobase by the photocatalyst, followed by hydrogen atom transfer from a thiol. The saturation of the C5-C6 backbone permits decarboxylation of the nonaromatic intermediate, and hydrolysis of the N4-amine constitutes a conversion from a cytosine derivative to a T-like base. This conversion demonstrates selectivity for 5-carboxycytosine over other canonical or modified nucleoside monomers, and is thereby applied to the sequencing of 5-carboxycytosine within modified oligonucleotides. The photochemistry explored in this study can also be used in conjunction with enzymatic oxidation by TET to profile 5-methylcytosine at single-base resolution. Compared to other base-conversion treatments, the rapid photochemical reaction takes place within minutes, which could provide advantages for high-throughput detection and diagnostic applications.

Selective Functionalisation of 5-Methylcytosine by Organic Photoredox Catalysis.

The epigenetic modification 5-methylcytosine plays a vital role in development, cell specific gene expression and disease states. The selective chemical modification of the 5-methylcytosine methyl group is challenging. Currently, no such chemistry exists. Direct functionalisation of 5-methylcytosine would improve the detection and study of this epigenetic feature. We report a xanthone-photosensitised process that introduces a 4-pyridine modification at a C(sp3 )-H bond in the methyl group of 5-methylcytosine. We propose a reaction mechanism for this type of reaction based on density functional calculations and apply transition state analysis to rationalise differences in observed reaction efficiencies between cyanopyridine derivatives. The reaction is initiated by single electron oxidation of 5-methylcytosine followed by deprotonation to generate the methyl group radical. Cross coupling of the methyl radical with 4-cyanopyridine installs a 4-pyridine label at 5-methylcytosine. We demonstrate use of the pyridination reaction to enrich 5-methylcytosine-containing ribonucleic acid.

Simultaneous sequencing of genetic and epigenetic bases in DNA.

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.

Chem-map profiles drug binding to chromatin in cells.

Characterizing drug-target engagement is essential to understand how small molecules influence cellular functions. Here we present Chem-map for in situ mapping of small molecules that interact with DNA or chromatin-associated proteins, utilizing small-molecule-directed transposase Tn5 tagmentation. We demonstrate Chem-map for three distinct drug-binding modalities as follows: molecules that target a chromatin protein, a DNA secondary structure or that intercalate in DNA. We map the BET bromodomain protein-binding inhibitor JQ1 and provide interaction maps for DNA G-quadruplex structure-binding molecules PDS and PhenDC3. Moreover, we determine the binding sites of the widely used anticancer drug doxorubicin in human leukemia cells; using the Chem-map of doxorubicin in cells exposed to the histone deacetylase inhibitor tucidinostat reveals the potential clinical advantages of this combination therapy. In situ mapping with Chem-map of small-molecule interactions with DNA and chromatin proteins provides insights that will enhance understanding of genome and chromatin function and therapeutic interventions.

Selective Functionalisation of 5-Methylcytosine by Organic Photoredox Catalysis.

The epigenetic modification 5-methylcytosine plays a vital role in development, cell specific gene expression and disease states. The selective chemical modification of the 5-methylcytosine methyl group is challenging. Currently, no such chemistry exists. Direct functionalisation of 5-methylcytosine would improve the detection and study of this epigenetic feature. We report a xanthone-photosensitised process that introduces a 4-pyridine modification at a C(sp3)-H bond in the methyl group of 5-methylcytosine. We propose a reaction mechanism for this type of reaction based on density functional calculations and apply transition state analysis to rationalise differences in observed reaction efficiencies between cyanopyridine derivatives. The reaction is initiated by single electron oxidation of 5-methylcytosine followed by deprotonation to generate the methyl group radical. Cross coupling of the methyl radical with 4-cyanopyridine installs a 4-pyridine label at 5-methylcytosine. We demonstrate use of the pyridination reaction to enrich 5-methylcytosine-containing ribonucleic acid.

DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody.

G-quadruplexes (G4s) are four-stranded DNA secondary structures that occur in the human genome and play key roles in transcription, replication, and genome stability. G4-specific molecular probes are of vital importance to elucidate the structure and function of G4s. The scFv antibody BG4 has been a widely used G4 probe but has various limitations, including relatively poor in vitro expression and the inability to be expressed intracellularly to interrogate G4s in live cells. To address these considerations, we describe herein the development of SG4, a camelid heavy-chain-only derived nanobody that was selected against the human Myc DNA G4 structure. SG4 exhibits low nanomolar affinity for a wide range of folded G4 structures in vitro. We employed AlphaFold combined with molecular dynamics simulations to construct a molecular model for the G4-nanobody interaction. The structural model accurately explains the role of key amino acids and Kd measurements of SG4 mutants, including arginine-to-alanine point mutations that dramatically diminish G4 binding affinity. Importantly, predicted amino acid-G4 interactions were subsequently confirmed experimentally by biophysical measurements. We demonstrate that the nanobody can be expressed intracellularly and used to image endogenous G4 structures in live cells. We also use the SG4 protein to positionally map G4s in situ and also on fixed chromatin. SG4 is a valuable, new tool for G4 detection and mapping in cells.

Self-organization of plasticity and specialization in a primitively social insect.

Biological systems have the capacity to not only build and robustly maintain complex structures but also to rapidly break up and rebuild such structures. Here, using primitive societies of Polistes wasps, we show that both robust specialization and rapid plasticity are emergent properties of multi-scale dynamics. We combine theory with experiments that, after perturbing the social structure by removing the queen, correlate time-resolved multi-omics with video recordings. We show that the queen-worker dimorphism relies on the balance between the development of a molecular queen phenotype in all insects and colony-scale inhibition of this phenotype via asymmetric interactions. This allows Polistes to be stable against intrinsic perturbations of molecular states while reacting plastically to extrinsic cues affecting the whole society. Long-term stability of the social structure is reinforced by dynamic DNA methylation. Our study provides a general principle of how both specialization and plasticity can be achieved in biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Early detection of cancer.

Survival improves when cancer is detected early. However, ~50% of cancers are at an advanced stage when diagnosed. Early detection of cancer or precancerous change allows early intervention to try to slow or prevent cancer development and lethality. To achieve early detection of all cancers, numerous challenges must be overcome. It is vital to better understand who is at greatest risk of developing cancer. We also need to elucidate the biology and trajectory of precancer and early cancer to identify consequential disease that requires intervention. Insights must be translated into sensitive and specific early detection technologies and be appropriately evaluated to support practical clinical implementation. Interdisciplinary collaboration is key; advances in technology and biological understanding highlight that it is time to accelerate early detection research and transform cancer survival.

Is there a role for upper gastrointestinal contrast study to predict the outcomes of sleeve gastrectomy? Lessons learnt from a prospective study.

CONTEXT: : The importance of upper gastrointestinal (UGI) contrast study following sleeve gastrectomy (SG) is equivocal. It can, however, yield anatomical and functional details, the significance of which mostly remains unknown. SETTINGS AND DESIGN: : This prospective, single-center study included SG patients between January 2018 and January 2019. MATERIALS AND METHODS: : UGI contrast study was done on post-operative day 1. The findings of the study namely gastroesophageal junction (GEJ) holdup time, presence of fundus, gastroduodenal emptying (GDE) time, and sleeve shape were compared with weight loss, improvement of glycosylated hemoglobin (HbA1c) and gastroesophageal reflux disease (GERD) symptoms at 3, 6, and 12 months follow-up. RESULTS: : There were 138 patients with 100% follow-up. Radiological sleeve patterns observed were: tubular (62.3%), superior (16.0%), and inferior (21.7%) pouches. GEJ holdup time had no effect on percentage total weight loss (%TWL) (P = 0.09) or HbA1c improvement (P = 0.077). The absence of fundus led to greater %TWL at 6 months (P = 0.048). GDE time <15 s led to higher %TWL (P = 0.028) and lower HbA1c (P = 0.010) at 12 months. Antrum size <2 cm was associated with higher %TWL (P = 0.022) and lower HbA1c level (P = 0.047) at 12 months. Vomiting and regurgitation were common with tubular sleeves. CONCLUSION: UGI contrast study can predict weight loss, HbA1c improvement, and GERD symptoms. The absence of fundus, small antrum, and rapid GDE are associated with better weight loss. HbA1c improvement is better with small antrum and rapid GDE. Tubular sleeve predisposes to vomiting and regurgitation.

G-quadruplex DNA structures in human stem cells and differentiation.

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.

Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm). How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting. Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.