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

Funder: Herchel Smith FundsThe 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

DNA G-quadruplex structures mould 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.This work is supported by a core CRUK award (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z). JS is a Marie Curie Fellow of the European Union (747297-QAPs-H2020-MSCA-IF-2016)

Using the affective priming paradigm to explore the attitudes underlying walking behaviour

Objectives. Walking is poorly represented in memory, making it difficult to measure using self-report and even harder to predict. To circumvent this, we used the affective priming paradigm (Fazio, Sanbonmatsu, Powell, & Kardes, 1986) to assess implicit attitudes towards walking. Methods. Royal Air Force trainee aircraftsmen (N ¼ 188) wore pedometers for 1 week prior to completing the affective priming paradigm, questionnaire and interview. The affective priming paradigm involved a computer-based response latency task containing physical activity words as primes followed by adjectives as targets to be evaluated. Targets were drawn from two bipolar dichotomies, good–bad (the original Fazio et al. items) and happy–sad (mood). Results. Priming for mood items was related to levels of physical activity with high frequency participants priming for the positive (happy) pole and low frequency participants priming for the negative (sad). Both groups primed for the negative element of the Fazio (good–bad) dichotomy. Regarding walking and running, there was no differentiation on the basis of participation level. Instead, facilitated responses to happy targets contrasted with inhibited responses to sad targets for both types of locomotion. There was weak evidence that intentions to run were associated with priming of positive target items, irrespective of category. Conclusions. The relationship between implicit attitudes and behaviour is complex. Whereas implicit attitudes were related to overall exercise participation, they were not related to the specific activity of walking, despite the behaviour being mainly under automatic control.</p

G-quadruplexes are transcription factor binding hubs in human chromatin

Abstract: 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

G-quadruplexes are transcription factor binding hubs in human chromatin

Abstract: 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

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

Funder: Herchel Smith FundsAbstract: 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

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis

Landscape of G-quadruplex DNA structural regions in breast cancer.

Response and resistance to anticancer therapies vary due to intertumor and intratumor heterogeneity1. Here, we map differentially enriched G-quadruplex (G4) DNA structure-forming regions (∆G4Rs) in 22 breast cancer patient-derived tumor xenograft (PDTX) models. ∆G4Rs are associated with the promoters of highly amplified genes showing high expression, and with somatic single-nucleotide variants. Differences in ΔG4R landscapes reveal seven transcription factor programs across PDTXs. ∆G4R abundance and locations stratify PDTXs into at least three G4-based subtypes. ∆G4Rs in most PDTXs (14 of 22) were found to associate with more than one breast cancer subtype, which we also call an integrative cluster (IC)2. This suggests the frequent coexistence of multiple breast cancer states within a PDTX model, the majority of which display aggressive triple-negative IC10 gene activity. Short-term cultures of PDTX models with increased ∆G4R levels are more sensitive to small molecules targeting G4 DNA. Thus, G4 landscapes reveal additional IC-related intratumor heterogeneity in PDTX biopsies, improving breast cancer stratification and potentially identifying new treatment strategies.The Caldas and Balasubramanian laboratories are supported by core funding from Cancer Research UK (C14303/A17197). The Balasubramanian laboratory is supported by Program grant funding from Cancer Research UK (C9681/A18618 and C9681/A29214) and a Wellcome Trust Investigator Award (209441/z/17/z). Prior to the revision of this study work by Dr. Robert Hänsel-Hertsch was supported by the Balasubramanian group, afterwards additionally supported by core funding of the Center for Molecular Medicine Cologne (CMMC)