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Unravelling the genomic targets of small molecules using high-throughput sequencing

Key Points

  • Small molecules can target protein and nucleic acid components of chromatin at specific genomic sites and perturb cellular processes.

  • Click chemistry relies on bio-orthogonal chemical reactivity that enables the introduction of a fluorophore to visualize small molecules in cells or the introduction of an affinity reagent that can be used for the purpose of target isolation.

  • High-throughput sequencing can be used to identify where small molecules influence the genome, which in some cases has provided new insights into drug responses.

  • Small molecules can be functionalized with affinity reagents to allow the isolation of DNA and characterization of genomic target sites by means of deep sequencing in a protocol known as Chem–seq.

  • A combination of experimental approaches — including ChIP–seq, Chem–seq and genome-wide gene expression analysis — can be used to delineate genome targeting with small molecules and might be useful for predicting cellular responses in the context of personalized medicine.

  • Chromatin influences genomic targeting with small molecules, thereby providing the opportunity for epigenome-targeting drugs to regulate and potentially reprogramme the response of certain drugs that operate at the genomic level.

Abstract

Small molecules — including various approved and novel cancer therapeutics — can operate at the genomic level by targeting the DNA and protein components of chromatin. Emerging evidence suggests that functional interactions between small molecules and the genome are non-stochastic and are influenced by a dynamic interplay between DNA sequences and chromatin states. The establishment of genome-wide maps of small-molecule targets using unbiased methodologies can help to characterize and exploit drug responses. In this Review, we discuss how high-throughput sequencing strategies, such as ChIP–seq (chromatin immunoprecipitation followed by sequencing) and Chem–seq (chemical affinity capture and massively parallel DNA sequencing), are enabling the comprehensive identification of small-molecule target sites throughout the genome, thereby providing insights into unanticipated drug effects.

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Figure 1: Targeting chromatin with small molecules.
Figure 2: Strategies for identifying genomic targets such as G4 DNA.
Figure 3: Genome-wide mapping of small-molecule target sites by Chem–seq.
Figure 4: Antibody-based versus small-molecule-based genomic target identification strategies.
Figure 5: Genome-wide strategies for identifying targets and responses of small molecules.
Figure 6: Influence of chromatin and chromatin modulators on genome targeting by small molecules.

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Acknowledgements

R.R. is supported by the French National Centre for Scientific Research (CNRS). K.M.M.'s laboratory is supported by start-up funds from University of Texas at Austin, USA, and by the Cancer Prevention Research Institute of Texas (CPRIT, R116). K.M.M. is a CPRIT Scholar in Cancer Research. The authors thank B. Xhemalce, M. Dawson and members of K.M.M.'s laboratory for critical reading of the manuscript.

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PowerPoint slides

Glossary

Chromatin

Nucleoprotein complex that packages and controls accessibility of the genome, thereby regulating transcription, DNA replication and repair.

Nucleosome

The basic unit of chromatin composed of ~147 base pairs of DNA wrapped around an octamer containing two copies of four core histone proteins (H2A, H2B, H3 and H4).

Post-translational modifications

(PTMs). Chemical modifications of, and ligation of small proteins (for example, ubiquitin and SUMO) on, substrate proteins that can serve as platforms for the binding of other proteins containing a 'reader' domain for specific PTMs.

Epigenome

Combination of chemical alterations of DNA, histone post-translational modifications and their interacting proteins that regulate genome accessibility and function.

Epigenetic

Pertaining to heritable phenotypic changes that are independent of alterations in the DNA sequence. A more general definition includes chromatin-mediated processes that are reliant on post-translational modifications of histone proteins, DNA methylation, histone variants or non-coding RNAs.

G-quadruplex

(G4). Alternative, non-Watson–Crick nucleic acid structures that arise from particular G-rich sequences. Guanine residues interact with one another within the same strand by means of hydrogen bonding and stacking interactions to produce four-stranded higher-order architectures. A broader definition also includes intermolecular structures formally composed of more than one strand.

Epigenetic modifier proteins

Proteins that 'write', 'erase' or 'read' epigenetic marks on chromatin.

Next-generation sequencing

(Also known as massively parallel sequencing). Post-Sanger sequencing techniques that can generate a DNA sequence from a single molecule of DNA rather than from multiple DNA templates, allowing millions of DNA fragments to be sequenced at the same time from a single sample.

ChIP–seq

(Chromatin immunoprecipitation followed by sequencing). An unbiased molecular biology-based protocol designed to identify interaction sites of chromatin-binding proteins with the genome. This method uses specific antibodies to precipitate the protein of interest, which isolates bound DNA fragments that are then subjected to sequencing.

Chem–seq

(Chemical affinity capture and massively parallel DNA sequencing). An unbiased molecular biology-based protocol designed to identify sites of interaction of small molecules with the genome. This method involves the use of small molecules of interest to isolate bound DNA targets, which are then analysed by sequencing.

Click chemistry

Selective, high-yielding and biocompatible chemical reactions used to covalently link two or more molecules. The copper-catalysed azide–alkyne cycloaddition (CuAAC) and its copper-free version are the most commonly used.

Topoisomerase 2

(TOP2). An enzyme that regulates DNA topology by cutting a pair of strands from a DNA helix to allow another unbroken helix to pass through, followed by resealing of the broken ends. TOP2 activity is essential for many DNA transactions and represents the target of several chemotherapeutic drugs.

CATCH-IT

(Covalent attachment of tagged histones to capture and identify turnover). A method for measuring genome-wide nucleosome turnover using metabolically labelled, newly synthesized histones for affinity-based chromatin capture.

FAIRE–seq

(Formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing). A method for identifying DNA from nucleosome-free regions.

Therapy-related secondary malignancies

Malignancies triggered by cancer treatment, including therapy-related myelodysplastic syndrome and acute myeloid leukaemia. These diseases have been linked to treatments with topoisomerase inhibitors. Potential causes include mutations such as chromosomal translocations that result from the inappropriate repair of treatment-induced DNA breaks.

Genomic imprinting

An epigenetic phenomenon that results in the monoallelic expression of a gene due to parental-dependent marking of the gene by DNA methylation or other epigenetic mechanisms.

TOP1

An enzyme that regulates DNA topology by breaking and rejoining a single strand from a DNA helix. TOP1 activity is essential for many DNA processes, including transcription and DNA replication, and is the target of several chemotherapeutic drugs.

Super enhancers

Specialized large cis-regulatory regions identified in embryonic stem cells and cancer cells that regulate the principal genes involved in cell identity and disease.

Personalized medicine

Customized health care in which clinical treatments are tailored to the patient. Diagnostic evaluation based on genetic and epigenetic information can be exploited in that context to select appropriate therapies.

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Rodriguez, R., Miller, K. Unravelling the genomic targets of small molecules using high-throughput sequencing. Nat Rev Genet 15, 783–796 (2014). https://doi.org/10.1038/nrg3796

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