The regulation and functions of DNA and RNA G-quadruplexes.

DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In this Review, we first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. We then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, we consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, we discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.

Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:

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.

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.

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.

Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts.

RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed knowledge of rG4-protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5'-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5'-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure-protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.

Identification of the extracellular matrix protein Fibulin-2 as a regulator of spinal nerve organization.

During amniote peripheral nervous system development, segmentation ensures the correct patterning of the spinal nerves relative to the vertebral column. Along the antero-posterior (rostro-caudal) axis, each somite-derived posterior half-sclerotome expresses repellent molecules to restrict axon growth and neural crest migration to the permissive anterior half-segment. To identify novel regulators of spinal nerve patterning, we investigated the differential gene expression of anterior and posterior half-sclerotomes in the chick embryo by RNA-sequencing. Several genes encoding extracellular matrix proteins were found to be enriched in either anterior (e.g. Tenascin-C, Laminin alpha 4) or posterior (e.g. Fibulin-2, Fibromodulin, Collagen VI alpha 2) half-sclerotomes. Among them, the extracellular matrix protein Fibulin-2 was found specifically restricted to the posterior half-sclerotome. By using in ovo ectopic expression in chick somites, we found that Fibulin-2 modulates spinal axon growth trajectories in vivo. While no intrinsic axon repellent activity of Fibulin-2 was found, we showed that it enhances the growth cone repulsive activity of Semaphorin 3A in vitro. Some molecules regulating axon growth during development are found to be upregulated in the adult central nervous system (CNS) following traumatic injury. Here, we found increased Fibulin-2 protein levels in reactive astrocytes at the lesion site of a mouse model of CNS injury. Together, these results suggest that the developing vertebral column and the adult CNS share molecular features that control axon growth and plasticity, which may open up the possibility for the identification of novel therapeutic targets for brain and spinal cord injury.

DSBCapture: in situ capture and sequencing of DNA breaks.

Double-strand DNA breaks (DSBs) continuously arise and cause mutations and chromosomal rearrangements. Here, we present DSBCapture, a sequencing-based method that captures DSBs in situ and directly maps these at single-nucleotide resolution, enabling the study of DSB origin. DSBCapture shows substantially increased sensitivity and data yield compared with other methods. Using DSBCapture, we uncovered a striking relationship between DSBs and elevated transcription within nucleosome-depleted chromatin.

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome.

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

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 as a molecular target for induced synthetic lethality in cancer cells.

Synthetic lethality is a genetic concept in which cell death is induced by the combination of mutations in two sensitive genes, while mutation of either gene alone is not sufficient to affect cell survival. Synthetic lethality can also be achieved "chemically" by combination of drug-like molecules targeting distinct but cooperative pathways. Previously, we reported that the small molecule pyridostatin (PDS) stabilizes G-quadruplexes (G4s) in cells and elicits a DNA damage response by causing the formation of DNA double strand breaks (DSB). Cell death mediated by ligand-induced G4 stabilization can be potentiated in cells deficient in DNA damage repair genes. Here, we demonstrate that PDS acts synergistically both with NU7441, an inhibitor of the DNA-PK kinase crucial for nonhomologous end joining repair of DNA DSBs, and BRCA2-deficient cells that are genetically impaired in homologous recombination-mediated DSB repair. G4 targeting ligands have potential as cancer therapeutic agents, acting synergistically with inhibition or mutation of the DNA damage repair machinery.

Mutations in FLVCR2 are associated with proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (Fowler syndrome).

Proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (PVHH), also known as Fowler syndrome, is an autosomal-recessively inherited prenatal lethal disorder characterized by hydranencephaly; brain stem, basal ganglia, and spinal cord diffuse clastic ischemic lesions with calcifications; glomeruloid vasculopathy of the central nervous system and retinal vessels; and a fetal akinesia deformation sequence (FADS) with muscular neurogenic atrophy. To identify the molecular basis for Fowler syndrome, we performed autozygosity mapping studies in three consanguineous families. The results of SNP microarrays and microsatellite marker genotyping demonstrated linkage to chromosome 14q24.3. Direct sequencing of candidate genes within the target interval revealed five different germline mutations in FLVCR2 in five families with Fowler syndrome. FLVCR2 encodes a transmembrane transporter of the major facilitator superfamily (MFS) hypothesized to be involved in regulation of growth, calcium exchange, and homeostasis. This is the first gene to be associated with Fowler syndrome, and this finding provides a basis for further studies to elucidate the pathogenetic mechanisms and phenotypic spectrum of associated disorders.

Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease.

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.

An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2.

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.

The generation of vertebral segmental patterning in the chick embryo.

We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline.

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.

Single-cell mapping of DNA G-quadruplex structures in human cancer cells.

G-quadruplexes (G4s) are four-stranded DNA secondary structures that form in guanine-rich regions of the genome. G4s have important roles in transcription and replication and have been implicated in genome instability and cancer. Thus far most work has profiled the G4 landscape in an ensemble of cell populations, therefore it is critical to explore the structure-function relationship of G4s in individual cells to enable detailed mechanistic insights into G4 function. With standard ChIP-seq methods it has not been possible to determine if G4 formation at a given genomic locus is variable between individual cells across a population. For the first time, we demonstrate the mapping of a DNA secondary structure at single-cell resolution. We have adapted single-nuclei (sn) CUT&Tag to allow the detection of G4s in single cells of human cancer cell lines. With snG4-CUT&Tag, we can distinguish cellular identity from a mixed cell-type population solely based on G4 features within individual cells. Our methodology now enables genomic investigations on cell-to-cell variation of a DNA secondary structure that were previously not possible.

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.

Promoter G-quadruplex folding precedes transcription and is controlled by chromatin.

BACKGROUND: Four-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state. RESULTS: Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction. CONCLUSIONS: Overall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription.

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.