ABSTRACT Cell type-specific gene expression patterns and dynamics during development or in disease are controlled by _cis_-regulatory elements (CREs), such as promoters and enhancers.

Distinct classes of CREs can be characterized by their epigenomic features, including DNA methylation, chromatin accessibility, combinations of histone modifications and conformation of

local chromatin. Tremendous progress has been made in cataloguing CREs in the human genome using bulk transcriptomic and epigenomic methods. However, single-cell epigenomic and multi-omic

technologies have the potential to provide deeper insight into cell type-specific gene regulatory programmes as well as into how they change during development, in response to environmental

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OTHERS INTERPRETING NON-CODING DISEASE-ASSOCIATED HUMAN VARIANTS USING SINGLE-CELL EPIGENOMICS Article 09 May 2023 SINGLE-CELL MULTI-OME REGRESSION MODELS IDENTIFY FUNCTIONAL AND

DISEASE-ASSOCIATED ENHANCERS AND ENABLE CHROMATIN POTENTIAL ANALYSIS Article Open access 21 March 2024 LOW INPUT CAPTURE HI-C (LICHI-C) IDENTIFIES PROMOTER-ENHANCER INTERACTIONS AT

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ACKNOWLEDGEMENTS We apologize to those authors whose work we fail to include in this Review due to space constraints. Research in the Ren lab is funded by the Ludwig Institute and the

National Institutes of Health (NIH) grants 1UM1HG009402, 1U19MH114831, 1U01MH121282, 1R01AG066018, R01AG067153, U01DA052769, 1UM1HG011585, RF1MH124612, 1R56AG069107, R01EY031663,

1U01HG012059, R24AG073198 and RF1MH128838. The Center for Epigenomics was supported, in part, by the UC San Diego School of Medicine and by NIH grants R01EY030591, U01HL148867, U01DK120429

and R01HD102534. The Gaulton lab is funded by NIH grants DK114650, DK120429, DK122607, DK105554 and HG012059. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Center for Epigenomics, University

of California San Diego, La Jolla, CA, USA Sebastian Preissl & Bing Ren * Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of

Freiburg, Freiburg, Germany Sebastian Preissl * Department of Paediatrics, Paediatric Diabetes Research Center, University of California San Diego, La Jolla, CA, USA Kyle J. Gaulton *

Department of Cellular and Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, CA, USA Bing Ren * Ludwig Institute for Cancer Research, La Jolla, CA, USA

Bing Ren Authors * Sebastian Preissl View author publications You can also search for this author inPubMed Google Scholar * Kyle J. Gaulton View author publications You can also search for

this author inPubMed Google Scholar * Bing Ren View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS The authors contributed equally to all

aspects of the article. CORRESPONDING AUTHORS Correspondence to Sebastian Preissl, Kyle J. Gaulton or Bing Ren. ETHICS DECLARATIONS COMPETING INTERESTS B.R. is a shareholder and consultant

of Arima Genomics, Inc., and a co-founder of Epigenome Technologies, Inc. K.J.G. is a consultant of Genentech and a shareholder in Vertex Pharmaceuticals and Neurocrine Biosciences. These

relationships have been disclosed to and approved by the UCSD Independent Review Committee. S.P. declares no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Reviews

Genetics_ thanks T. Stuart, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS CHROMIUM SINGLE CELL MULTIOME ATAC+GENE EXPRESSION:

https://www.10xgenomics.com/products/single-cell-multiome-atac-plus-gene-expression SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION GLOSSARY * _Cis-_regulatory elements (CREs).

Non-coding DNA sequences that regulate transcription of genes located on the same chromosome. They include enhancers, promoters, insulators, silencing elements and tethering elements.

Different classes of CREs can be identified using a combination of molecular markers, including chromatin accessibility and epigenetic modifications. * Promoters CREs located at the

transcriptional start site of a gene. * Enhancers CREs that can activate target gene expression from a large genomic distance, ranging from several kilobases to even millions of base pairs.

They can be found either upstream or downstream of the target gene promoter. * Insulators CREs that prevent an enhancer from activating a target gene when placed between the enhancer and

gene promoter but not when placed outside. An insulator also refers to a boundary element that can prevent the spreading of heterochromatin into euchromatic regions. * Silencer elements CREs

that can be located close or distal to the transcriptional start site of the target gene. Silencers are bound by repressive transcription factors to inactivate gene expression. * Tethering

elements CREs that can bring together promoters and enhancers for gene activation. * Chromatin A complex of DNA and histone proteins. The basic unit of chromatin is the nucleosome. * Histone

modifications Covalent modifications to histone proteins, such as methylation, acetylation, phosphorylation, ubiquitylation and sumoylation, that take place at lysine, serine, threonine,

arginine and other residues. Histone modifications are catalysed by a diverse panel of enzymes referred to as writers, removed by a different set of proteins known as erasers, and recognized

by chromatin-binding proteins known as readers. Activity of CREs is directly linked to distinct histone modifications due to the activities of writers, erasers and readers. * Epigenome The

combined features that enable stable propagation of different gene expression patterns from the same genome sequence. These include methylation of DNA at cytosine bases (mC), chemical

modification of the histone proteins, chromatin accessibility and higher-order chromatin structures. * Tagmentation The process by which double-stranded DNA is cleaved by the transposase

Tn5, creating short DNA fragments that are simultaneously tagged with PCR adapters. Tagmentation using Tn5 preferentially occurs at accessible or open chromatin and this property is used in

ATAC-seq and other related assays. * 3D-chromatin organization Folding of the chromatin fibres inside the nucleus governs the spatial proximity between genes and CREs. While complex and

variable between cells, the chromatin organization exhibits certain common features, including A/B compartments, topologically associating domains and loops. RIGHTS AND PERMISSIONS Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Preissl, S., Gaulton, K.J. & Ren, B. Characterizing _cis_-regulatory elements using single-cell epigenomics. _Nat Rev Genet_ 24,

21–43 (2023). https://doi.org/10.1038/s41576-022-00509-1 Download citation * Accepted: 24 May 2022 * Published: 15 July 2022 * Issue Date: January 2023 * DOI:

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