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.

A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

Genome surveillance by HUSH-mediated silencing of intronless mobile elements.

All life forms defend their genome against DNA invasion. Eukaryotic cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 lysine 9 trimethylation (H3K9me3)1-3. How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. Here we show that HUSH is able to recognize and transcriptionally repress a broad range of long, intronless transgenes. Intron insertion into HUSH-repressed transgenes counteracts repression, even in the absence of intron splicing. HUSH binds transcripts from the target locus, prior to and independent of H3K9me3 deposition, and target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Genomic data reveal how HUSH binds and represses a subset of endogenous intronless genes generated through retrotransposition of cellular mRNAs. Thus intronless cDNA-the hallmark of reverse transcription-provides a versatile way to distinguish invading retroelements from host genes and enables HUSH to protect the genome from 'non-self' DNA, despite there being no previous exposure to the invading element. Our findings reveal the existence of a transcription-dependent genome-surveillance system and explain how it provides immediate protection against newly acquired elements while avoiding inappropriate repression of host genes.

RNA G-quadruplex structures control ribosomal protein production.

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5' UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells.

DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

G-quadruplexes are transcription factor binding hubs in human chromatin.

BACKGROUND: The binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed. RESULTS: Herein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes. CONCLUSIONS: Our results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription.

The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr.

Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.

ELIXIR and Toxicology: a community in development.

Toxicology has been an active research field for many decades, with academic, industrial and government involvement. Modern omics and computational approaches are changing the field, from merely disease-specific observational models into target-specific predictive models. Traditionally, toxicology has strong links with other fields such as biology, chemistry, pharmacology and medicine. With the rise of synthetic and new engineered materials, alongside ongoing prioritisation needs in chemical risk assessment for existing chemicals, early predictive evaluations are becoming of utmost importance to both scientific and regulatory purposes. ELIXIR is an intergovernmental organisation that brings together life science resources from across Europe. To coordinate the linkage of various life science efforts around modern predictive toxicology, the establishment of a new ELIXIR Community is seen as instrumental. In the past few years, joint efforts, building on incidental overlap, have been piloted in the context of ELIXIR. For example, the EU-ToxRisk, diXa, HeCaToS, transQST, and the nanotoxicology community have worked with the ELIXIR TeSS, Bioschemas, and Compute Platforms and activities. In 2018, a core group of interested parties wrote a proposal, outlining a sketch of what this new ELIXIR Toxicology Community would look like. A recent workshop (held September 30th to October 1st, 2020) extended this into an ELIXIR Toxicology roadmap and a shortlist of limited investment-high gain collaborations to give body to this new community. This Whitepaper outlines the results of these efforts and defines our vision of the ELIXIR Toxicology Community and how it complements other ELIXIR activities.

Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids.

The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.

Genome-wide DNA Methylation Signatures Are Determined by DNMT3A/B Sequence Preferences.

Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.

Haemophilus influenzae Glucose Catabolism Leading to Production of the Immunometabolite Acetate Has a Key Contribution to the Host Airway-Pathogen Interplay.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo. Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease.

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus.

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress-associated sigma B factor (σB ). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σB activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs.

Genetic interactions of G-quadruplexes in humans.

G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.

Sequencing abasic sites in DNA at single-nucleotide resolution.

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.

All-Fullerene Electron Donor-Acceptor Conjugates.

The synthesis and characterization of a covalent all-fullerene C60 -Lu3 N@Ih -C80 electron donor-acceptor conjugate has been realized by sequential 1,3-dipolar cycloaddition reactions of azomethine ylides on Lu3 N@Ih -C80 and C60 . To the best of our knowledge, this is the first time that two fullerenes behaving as both electron donor (Lu3 N@Ih -C80 ) and acceptor (C60 ) are forming an electroactive dumbbell. DFT calculations reveal up to 16 diastereomeric pairs, that is, 8 with syn and 8 with anti orientation, with the anti-RSSS isomer being the most stable. Spectroelectrochemical absorption and femtosecond transient absorption experiments support the notion that a C60 ⋅- -Lu3 N@Ih -C80 ⋅+ charge-separated state is formed. Spin conversion from the charge-separated singlet state C60 ⋅- -Lu3 N@Ih -C80 ⋅+ into the corresponding triplet state is facilitated by the heavy-atom effect stemming from the Lu3 N-cluster, which, in turn, slows down the charge recombination by one order of magnitude.

5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells.

Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information. The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine (5fC) is linked to tissue-specific nucleosome organization. Our study reveals that 5fC is associated with increased nucleosome occupancy in vitro and in vivo. We demonstrate that 5fC-associated nucleosomes at enhancers in the mammalian hindbrain and heart are linked to elevated gene expression. Our study also reveals the formation of a reversible-covalent Schiff base linkage between lysines of histone proteins and 5fC within nucleosomes in a cellular environment. We define their specific genomic loci in mouse embryonic stem cells and look into the biological consequences of these DNA-histone Schiff base sites. Collectively, our findings show that 5fC is a determinant of nucleosome organization and plays a role in establishing distinct regulatory regions that control transcription.

DNA G-quadruplex structures mold the DNA methylome.

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.

Base resolution maps reveal the importance of 5-hydroxymethylcytosine in a human glioblastoma.

Aberrant genetic and epigenetic variations drive malignant transformation and are hallmarks of cancer. Using PCR-free sample preparation we achieved the first in-depth whole genome (hydroxyl)-methylcytosine, single-base-resolution maps from a glioblastoma tumour/margin sample of a patient. Our data provide new insights into how genetic and epigenetic variations are interrelated. In the tumour, global hypermethylation with a depletion of 5-hydroxymethylcytosine was observed. The majority of single nucleotide variations were identified as cytosine-to-thymine deamination products within CpG context, where cytosine was preferentially methylated in the margin. Notably, we observe that cells neighbouring tumour cells display epigenetic alterations characteristic of the tumour itself although genetically they appear "normal". This shows the potential transfer of epigenetic information between cells that contributes to the intratumour heterogeneity of glioblastoma. Together, our reference (epi)-genome provides a human model system for future studies that aim to explore the link between genetic and epigenetic variations in cancer progression.