G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.
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.Nucleic acids have considerable potential to fold into three-dimensional, 'secondary' structures. This can happen through the formation of non-Watson-Crick hydrogen bonds between nucleobases. Early observations on the self-assembly of guanylic acid 1 led to the elucidation of the guanine tetrad-forming sequence motif 2 (FIG. 1a), in which guanines are mutually bonded by Hoogsteen hydrogen base-pairing to form a planar array that is further
It has been hypothesized that G-quadruplexes can sequester the 3′ end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures implies that parallel telomeric G-quadruplexes may play an important biological role.
Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.
The ability of most cancer cells to grow indefinitely relies on the enzyme telomerase and its recruitment to telomeres. In human cells, recruitment depends on the Cajal body RNA chaperone TCAB1 binding to the RNA subunit of telomerase (hTR) and is also thought to rely on an N-terminal domain of the catalytic subunit, hTERT. We demonstrate that coilin, an essential structural component of Cajal bodies, is required for endogenous telomerase recruitment to telomeres but that overexpression of telomerase can compensate for Cajal body absence. In contrast, recruitment of telomerase was sensitive to levels of TCAB1, and this was not rescued by overexpression of telomerase. Thus, although Cajal bodies are important for recruitment, TCAB1 has an additional role in this process that is independent of these structures. TCAB1 itself localizes to telomeres in a telomerase-dependent but Cajal body-independent manner. We identify a point mutation in hTERT that largely abolishes recruitment yet does not affect association of telomerase with TCAB1, suggesting that this region mediates recruitment by an independent mechanism. Our results demonstrate that telomerase has multiple independent requirements for recruitment to telomeres and that the function of TCAB1 is to directly transport telomerase to telomeres.T elomeres are protein-nucleic acid structures at the ends of linear chromosomes, which protect the DNA termini from degradation and inappropriate processing as damaged DNA. Despite this protective role, telomere shortening still occurs in most normal human somatic cells during DNA replication due to inherent limitations in the replication machinery, and this shortening is the basis of cellular senescence (5,14,28). Approximately 85 to 90% of human cancers counteract this shortening and avoid senescence by activating the ribonucleoprotein telomerase to extend telomeres (13,30). Active telomerase consists of three core components essential for activity (7): hTERT, the reverse transcriptase catalytic subunit (26); hTR, the RNA subunit, used as a cognate template for reverse transcription of telomeric DNA (12); and the protein dyskerin, which is essential for hTR stability (23). The extension of telomeres by telomerase is preceded by a complex series of events involving enzyme biogenesis, transport from sites of enzyme assembly, and trafficking of telomerase in the nucleus at the appropriate phase in the cell cycle. The factors involved in these steps and how these stages are integrated are not fully understood.Regions of hTERT have been identified that are essential for the enzyme to extend the life span of untransformed cells but which are dispensable for enzyme function ex vivo (2, 4). These regions, which separate the in vivo functionality of the enzyme from ex vivo telomerase activity, were termed DAT for "dissociates the activities of telomerase." A potential explanation for this observation is a failure of the enzyme to be transported or recruited to telomeres. Fusion of the single-stranded telomeric DNA binding protein...
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.
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.
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