A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity
Open accessAbstract
A gene's position in the genome can profoundly affect its expression because regional differences in chromatin modulate the activity of locally acting cis-regulatory sequences (CRSs). Here we study how CRSs and regional chromatin act in concert on a genome-wide scale. We present a massively parallel reporter gene assay that measures the activities of hundreds of different CRSs, each integrated at many specific genomic locations. Although genome location strongly affected CRS activity, the relative strengths of CRSs were maintained at all chromosomal locations. The intrinsic activities of CRSs also correlated with their activities in plasmid-based assays. We explain our data with a quantitative model in which expression levels are set by independent contributions from local CRSs and the regional chromatin environment, rather than by more complex sequence- or protein-specific interactions between these two factors. The methods we present will help investigators determine when regulatory information is integrated in a modular fashion and when regulatory sequences interact in more complex ways.
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Enhancers are punctate relative to broader chromatin domains, but their in vivo functionality depends on chromatin context and the trans milieu.
“Enhancers are ‘punctate’ relative to broader chromatin domains (e.g. chromosomes, topologically associating domains [G] (TADs) and sub-compartments of TADs87), but their in vivo functionality is dependent on both the chromatin context in which they reside88 and on the trans milieu (e.g. cell-type-specific TFs)89.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [88]
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Ribeiro-dos-Santos, A. M., Hogan, M. S., Luther, R. D., Brosh, R., & Maurano, M. T. (2022) · Genome Research · doi:10.1101/gr.276449.121 · Cited by 14 · see citing works
“In contrast, integrated barcoded reporter assays (Akhtar et al. 2013; Maricque et al. 2018; Moudgil et al. 2020) offer the potential to directly assess the interaction between novel CTCF sites and the endogenous genomic landscape.”
BackgroundDecoding polygenic diseases: advances in noncoding variant prioritization and validation
Chin, I. M., Gardell, Z. A., & Corces, M. R. (2024) · Trends in Cell Biology · doi:10.1016/j.tcb.2024.03.005 · Cited by 16 · see citing works
Just a SNP away: The future of in vivo massively parallel reporter assay
Degner, K. N., Bell, J., Jones, S. D., & Won, H. (2024) · Cell Insight · doi:10.1016/j.cellin.2024.100214 · Cited by 13 · see citing works
Functional characterization of enhancer activity during a long terminal repeat's evolutionPreprint
Du, A. Y., Zhuo, X., Sundaram, V., Jensen, N. O., Chaudhari, H. G., Saccone, N. L., Cohen, B. A., & Wang, T. (2022) · Genome Research · doi:10.1101/gr.276863.122 · Cited by 14 · see citing works
Self-citationZheng, Y., & Chen, S. (2024) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2024.1347436 · Cited by 14 · see citing works
“Besides MPRAs, many assays have been developed to interrogate other aspects of transcription factor-gene expression relationships and can be readily adapted to the retinal system (Wang et al., 2012; Arnold et al., 2013; Dixit et al., 2016; Muraro et al., 2016; Maricque et al., 2019).”
MethodFrochaux, M., Sleiman, M. S. B., Gardeux, V., Dainese, R., Hollis, B., Litovchenko, M., Braman, V. S., Andreani, T., Osman, D., & Deplancke, B. (2020) · Genome biology · doi:10.1186/s13059-019-1912-z · Cited by 13 · see citing works
Strategies for dissecting the complexity of neurodevelopmental disorders
Sun, J., Noss, S., Banerjee, D., Das, M., & Girirajan, S. (2023) · Trends in Genetics · doi:10.1016/j.tig.2023.10.009 · Cited by 10 · see citing works
Massively parallel characterization of insulator activity across the genome
Hong, C. K., Wu, Y., Erickson, A., Li, J., Federico, A. J., & Cohen, B. A. (2024) · Nature Communications · doi:10.1038/s41467-024-52599-6 · Cited by 12 · see citing works
“All insulator sequences and mutants can be found in Supplementary Data 10 and individual gen-bank fi les are provided as Supplementary Data 11.”
BackgroundInfluentialSelf-citationLoci specific epigenetic drug sensitivity
Zhang, T., Pilko, A., & Wollman, R. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa210 · Cited by 12 · see citing works
“Complex sequence- or proteinspecific interactions between local chromatin and synthetic reporters were not observed in previous study as well (11).”
BackgroundMultiplex profiling of developmental enhancers with quantitative, single-cell expression reportersPreprint
Lalanne, J.-B., Regalado, S. G., Domcke, S., Calderon, D., Martin, B., Li, T., Suiter, C. C., Lee, C., Trapnell, C., & Shendure, J. (2022) · bioRxiv · doi:10.1101/2022.12.10.519236 · Cited by 14 · see citing works
Rapid and quantitative functional interrogation of human enhancer variant activity in live mice
Hollingsworth, E. W., Liu, T. A., Alcantara, J. A., Chen, C. X., Jacinto, S. H., & Kvon, E. Z. (2025) · Nature Communications · doi:10.1038/s41467-024-55500-7 · Cited by 11 · see citing works
“These cell-type-speci fi c effects of enhancer variants are dif fi cult to capture with high-throughput methods such as massively parallel reporter assays (MPRAs) and CRISPR inhibitor/activator screens, both of which are primarily performed in vitro 27,28 or in one tissue 29 – 34 .”
Alakuş, T. B. (2023) · Biomimetics · doi:10.3390/biomimetics8020218 · Cited by 10 · see citing works
“Although these enhancers are far from the transcription start point, they are close to each other in three-dimensional space [3].”
BackgroundFunctional genomic assays to annotate enhancer–promoter interactions genome wide
Leung, A. K.-Y., Li, Y., & Yu, H. (2022) · Human Molecular Genetics · doi:10.1093/hmg/ddac204 · Cited by 9 · see citing works
“In the future, a revisit of current conclusions will be of great importance with improved versions of in-genome ectopic reporter assays (63,64) that are capable of swapping both enhancers and promoters (Fig.”
BackgroundGödecke, N., Herrmann, S., Häuser, H., Mayer‐Bartschmid, A., Trautwein, M., & Wirth, D. (2020) · ACS Synthetic Biology · doi:10.1021/acssynbio.0c00519 · Cited by 9 · see citing works
Using individual barcodes to increase quantification power of massively parallel reporter assays
Keukeleire, P., Rosen, J. D., Göbel-Knapp, A., Salomon, K., Schubach, M., & Kircher, M. (2025) · BMC Bioinformatics · doi:10.1186/s12859-025-06065-9 · Cited by 8 · see citing works
Large-scale discovery of potent, compact and erythroid specific enhancers for gene therapy vectors
Psatha, N., Sova, P., Georgolopoulos, G., Paschoudi, K., Iwata, M., Bloom, J. E., Ulyanova, T., Wang, H., Kirtsou, A., Vasiloudis, N.-I., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-59235-x · Cited by 7 · see citing works
“Because expression of integrated lentiviral cas-settes is impacted by local genomic and epigenomic context (i.e. position-effect variegation) 16 , we assessed the number of unique integrations required for each element to yield reliable expression estimates.”
Chen, C., Wang, Z., Kang, M., Lee, K. B., & Ge, X. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad1001 · Cited by 7 · see citing works
Kribelbauer-Swietek, J. F., Gardeux, V., Llimos, G., Faltejsková, K., Russeil, J., Grenningloh, N., Levassor, L., Steiner, C., Vondrášek, J., & Deplancke, B. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.12.08.627402 · Cited by 6 · see citing works
Baderna, V., Barzaghi, G., Kleinendorst, R., Chatsirisupachai, K., Moniot-Perron, L., Doyle, C., Schopp, M., Hochepied, T., Libert, C., Odom, D. T., et al. (2026) · Nature Genetics · doi:10.1038/s41588-026-02703-x · Cited by 8 · see citing works
“Our data provide a potential molecular mechanism to explain the effects of the chromatin environment on enhancer activities 52–54 .”
Mulvey, B., Lagunas, T., & Dougherty, J. D. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.02.02.931337 · Cited by 4 · see citing works
Long-range massively parallel reporter assay reveals rules of distal enhancer-promoter interactionsPreprint
Wu, Y., Li, J., Bartley-Dier, E. L., Pitts, C., & Cohen, B. A. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.04.21.649048 · Cited by 5 · see citing works
“The promoter landing pad expresses the mEmerald fluorescent protein and iCaspase9 for counterselection and is flanked by loxFAS and loxP sites for orthogonal RMCE. b-c, Workflow of long-range MPRA. eBC: enhancer barcode; rBC: random barcode; dBC: distance barcode.”
Self-citationPopulation Diversity at the Single-Cell Level
Gordon, M. G., Kathail, P., Choy, B., Kim, M. C., Mazumder, T., Gearing, M., & Ye, C. (2024) · Annual Review of Genomics and Human Genetics · doi:10.1146/annurev-genom-021623-083207 · Cited by 4 · see citing works
Large-scale analysis of the integration of enhancer-enhancer signals by promoters
Martinez-Ara, M., Comoglio, F., & van Steensel, B. (2024) · eLife · doi:10.7554/elife.91994.3 · Cited by 4 · see citing works
Position Effect Variegation: Role of the Local Chromatin Context in Gene Expression Regulation
Boldyreva, L. V., Andreyeva, E. N., & Pindyurin, A. V. (2022) · Molecular Biology · doi:10.1134/s0026893322030049 · Cited by 3 · see citing works
Methods of massive parallel reporter assays for investigation of enhancers
Romanov, S. E., Kalashnikova, D. A., & Laktionov, P. P. (2021) · Vavilov Journal of Genetics and Breeding · doi:10.18699/vj21.038 · Cited by 3 · see citing works
“The approaches described above did not allow answering one of the fundamental questions of understanding the principles of enhancers’ activity, namely, to what extent is it determined by the DNA sequence, and to what extent – by the properties of the surrounding chromatin? A systematic study of this issue was carried…”
BackgroundMethodFehér, A., Schnúr, A., Muenthaisong, S., Bellák, T., Ayaydin, F., Várady, G., Kemter, E., Wolf, E., & Dinnyés, A. (2022) · Scientific Reports · doi:10.1038/s41598-022-12956-1 · Cited by 3 · see citing works
Cofactors: a new layer of specificity to enhancer regulation
Kreibich, E., & Krebs, A. R. (2022) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2022.07.008 · Cited by 3 · see citing works
Compatibility logic of human enhancer and promoter sequencesPreprint
Bergman, D. T., Jones, T., Liu, V., Siraj, L., Kang, H., Nasser, J., Kane, M., Nguyen, T. H., Grossman, S. R., Fulco, C. P., et al. (2021) · bioRxiv · doi:10.1101/2021.10.23.462170 · Cited by 5 · see citing works
Large-scale analysis of the integration of enhancer-enhancer signals by promotersPreprint
Martinez-Ara, M., Comoglio, F., & van Steensel, B. (2023) · eLife · doi:10.7554/elife.91994.1 · Cited by 3 · see citing works
Wang, Z., Sarkar, A., & Ge, X. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.11.27.625413 · Cited by 3 · see citing works
Cumulative TF binding and H3K27 Acetylation drive enhancer activation frequencyPreprint
Baderna, V., Barzaghi, G., Kleinendorst, R., Chatsirisupachai, K., Moniot-Perron, L., Schopp, M., Hochepied, T., Libert, C., Odom, D. T., Zaugg, J. B., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.03.26.645413 · Cited by 3 · see citing works
“Modulation of accessibility frequency by activating chromatin marks could account for enhancer activity shifts observed at various loci in multiplexed reporter assays (44–46).”
Human Accelerated Regions: Functional Studies and Methodological Approaches (A Review)
Knyazeva, A. S., & Шнайдер, Т. А. (2026) · Cell and Tissue Biology · doi:10.1134/s1990519x25600784 · Cited by 1 · see citing works
How to build the regulatory genome: a constructionist guide to the cis-regulatory code
Bell, C. C., de Boer, C. G., Camellato, B. R., Delàs, M. J., Ibrahim, D. M., & Montgomery, M. T. (2026) · Development · doi:10.1242/dev.205516 · Cited by 1 · see citing works
Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants
Kang, C.-K., & Kim, A.-R. (2023) · iScience · doi:10.1016/j.isci.2023.108747 · Cited by 1 · see citing works
Siegel, D. A., Tonquèze, O. L., Biton, A., Erle, D. J., & Zaitlen, N. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.02.12.945113 · Cited by 1 · see citing works
“MPRAs are now a fundamental tool and have been used to identify enhancer activity[1], determine the influence of chromatin on cis-regulatory sequences [2], identify sources of variation in red blood cell traits [3], and identify functional annotations in 3′ untranslated regions[4].”
MethodLarge-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectorsPreprint
Psatha, N., Sova, P., Georgolopoulos, G., Paschoudi, K., Iwata, M., Bloom, J. E., Ulyanova, T., Wang, H., Kirtsou, A., Vasiloudis, N.-I., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.10.04.559165 · Cited by 1 · see citing works
Large-scale analysis of the integration of enhancer-enhancer signals by promotersPreprint
Martinez-Ara, M., Comoglio, F., & van Steensel, B. (2024) · eLife · doi:10.7554/elife.91994.2 · Cited by 1 · see citing works
Sequence determinants and evolution of constitutive and alternative splicing in yeast speciesPreprint
Schirman, D., Zohar Yakhini, B., Dahan, O., & Pilpel, Y. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.04.20.050609
“2010), was used previously to explore different elements involved in regulation of transcription, translation, RNA stability, and other regulatory elements (Sharon et al. 2012; Kosuri et al. 2013; Shalem et al. 2015; Weingarten-Gabbay et al. 2016; Levy et al. 2017; Maricque et al. 2018).”
MethodRenganaath, K., Cheung, R., Day, L., Kosuri, S., Kruglyak, L., & Albert, F. (2020) · bioRxiv · doi:10.1101/2020.08.31.276337
“MPRAs have been conducted using plasmid-borne reporters as well as reporters integrated into the genome (Inoue et al., 2017; Maricque et al., 2019; Mogno et al., 2013).”
BackgroundMethodInfluentialModular effects of gene promoters and chromatin environments on noise in gene expressionPreprint
Zhao, S., Pincus, Z., & Cohen, B. A. (2021) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2021.04.29.441875
“K562 cell lines carrying landing pads constructions were taken from our previously published work (Maricque et al., 2018).”
BackgroundMethodInfluentialSelf-citationMefford, H. C. (2020) · Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · doi:10.3410/f.736437709.793571951
A DNA base-specific sequence interposed between CRX and NRL contributes to RHODOPSIN expression
Maritato, R., Medugno, A., D’Andretta, E., Riso, G. D., Lupo, M., Botta, S., Marrocco, E., Renda, M., Sofia, M., Mussolino, C., et al. (2024) · Scientific Reports · doi:10.1038/s41598-024-76664-8
“AAV vectors delivered DNA to the target tissue, including in the retina 71 , remains almost exclusively episomal 71,74,75 , thereby isolating cis -regulatory activity from the genome context 76,77 .”
BackgroundMethodRezaei, N., Dormiani, K., Kiani‐Esfahani, A., Mirdamadian, S., Rahmani, M., Jafarpour, F., & Nasr‐Esfahani, M. H. (2024) · Scientific Reports · doi:10.1038/s41598-024-77614-0
“It has been shown that enhancers respond differently to various types of promoters 58 .”
BackgroundSheng, Y., Tu, X., & Mostafavi, S. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.03.11.642630
“For datasets with FACS as the readout assay, the original bin indices were retained as output values and used as indicators of transcriptional efficiency 4,5,18 .”
Mechanisms and functional implications of long-range enhancer-dependent gene regulation
Iliadou, M., Haro, E., & Rada-Iglesias, Á. (2026) · Nature Genetics · doi:10.1038/s41588-026-02721-9
“However, episomal assays do not fully recapitulate chromatin context or genomic distance, both of which can strongly influence enhancer activity 15,17,49 .”
- Authors
- Maricque, B. B., Chaudhari, H. G., & Cohen, B. A.
- Affiliations
- Washington University in St. Louis (US)
- Corresponding author
- Cohen, Barak A
- Venue
- Nature Biotechnology
- Published
- 2018-11-19
- Volume
- 37
- Issue
- 1
- Pages
- 90-95
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC7351048
- OpenAlex
- W2900900797
- Cited by
- 83
- References
- 50
- Citations by year
- 2026: 6 · 2025: 5 · 2024: 16 · 2023: 11 · 2022: 15 · 2021: 6 · 2020: 16 · 2019: 7 · 2018: 1
- FWCI
- 2.62
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Alvin J. Siteman Cancer Center
- Hope Center for Neurological Disorders
- National Institutes of Health
- National Human Genome Research Institute (R01HG008687)
- National Cancer Institute (P30CA091842)
- National Institute of General Medical Sciences (R01GM092910)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- cis-regulatory sequences, chromatin structure, massively parallel reporter assay, gene expression regulation
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