Massively Parallel Reporter Perturbation Assays Uncover Temporal Regulatory Architecture During Neural Differentiation

Overview

Journal Nat Commun

Specialty Biology

Date 2022 Mar 22

PMID 35315433

Authors

Anat Kreimer

Tal Ashuach

Fumitaka Inoue

Alex Khodaverdian

Chengyu Deng

Nir Yosef

Nadav Ahituv

Affiliations

Soon will be listed here.

Abstract

Gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we perform perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineate four categories of functional elements, and observe that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

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References

Schoenfelder S, Fraser P . Long-range enhancer-promoter contacts in gene expression control. Nat Rev Genet. 2019; 20(8):437-455. DOI: 10.1038/s41576-019-0128-0. View

Maurano M, Humbert R, Rynes E, Thurman R, Haugen E, Wang H . Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012; 337(6099):1190-5. PMC: 3771521. DOI: 10.1126/science.1222794. View

Kearns N, Pham H, Tabak B, Genga R, Silverstein N, Garber M . Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods. 2015; 12(5):401-403. PMC: 4414811. DOI: 10.1038/nmeth.3325. View

Korkmaz G, Lopes R, Ugalde A, Nevedomskaya E, Han R, Myacheva K . Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol. 2016; 34(2):192-8. DOI: 10.1038/nbt.3450. View

Inoue F, Ahituv N . Decoding enhancers using massively parallel reporter assays. Genomics. 2015; 106(3):159-164. PMC: 4540663. DOI: 10.1016/j.ygeno.2015.06.005. View

Inoue F, Kircher M, Martin B, Cooper G, Witten D, McManus M . A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity. Genome Res. 2016; 27(1):38-52. PMC: 5204343. DOI: 10.1101/gr.212092.116. View

White M, Myers C, Corbo J, Cohen B . Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks. Proc Natl Acad Sci U S A. 2013; 110(29):11952-7. PMC: 3718143. DOI: 10.1073/pnas.1307449110. View

Kheradpour P, Ernst J, Melnikov A, Rogov P, Wang L, Zhang X . Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. Genome Res. 2013; 23(5):800-11. PMC: 3638136. DOI: 10.1101/gr.144899.112. View

Kwasnieski J, Fiore C, Chaudhari H, Cohen B . High-throughput functional testing of ENCODE segmentation predictions. Genome Res. 2014; 24(10):1595-602. PMC: 4199366. DOI: 10.1101/gr.173518.114. View

10.

Ulirsch J, Nandakumar S, Wang L, Giani F, Zhang X, Rogov P . Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits. Cell. 2016; 165(6):1530-1545. PMC: 4893171. DOI: 10.1016/j.cell.2016.04.048. View

11.

Wang X, He L, Goggin S, Saadat A, Wang L, Sinnott-Armstrong N . High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human. Nat Commun. 2018; 9(1):5380. PMC: 6300699. DOI: 10.1038/s41467-018-07746-1. View

12.

Soufi A, Donahue G, Zaret K . Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome. Cell. 2012; 151(5):994-1004. PMC: 3508134. DOI: 10.1016/j.cell.2012.09.045. View

13.

Yosef N, Shalek A, Gaublomme J, Jin H, Lee Y, Awasthi A . Dynamic regulatory network controlling TH17 cell differentiation. Nature. 2013; 496(7446):461-8. PMC: 3637864. DOI: 10.1038/nature11981. View

14.

Spitz F, Furlong E . Transcription factors: from enhancer binding to developmental control. Nat Rev Genet. 2012; 13(9):613-26. DOI: 10.1038/nrg3207. View

15.

Whitfield T, Wang J, Collins P, Partridge E, Force Aldred S, Trinklein N . Functional analysis of transcription factor binding sites in human promoters. Genome Biol. 2012; 13(9):R50. PMC: 3491394. DOI: 10.1186/gb-2012-13-9-r50. View

16.

Biggin M . Animal transcription networks as highly connected, quantitative continua. Dev Cell. 2011; 21(4):611-26. DOI: 10.1016/j.devcel.2011.09.008. View

17.

Landolin J, Johnson D, Trinklein N, Aldred S, Medina C, Shulha H . Sequence features that drive human promoter function and tissue specificity. Genome Res. 2010; 20(7):890-8. PMC: 2892090. DOI: 10.1101/gr.100370.109. View

18.

Grossman S, Zhang X, Wang L, Engreitz J, Melnikov A, Rogov P . Systematic dissection of genomic features determining transcription factor binding and enhancer function. Proc Natl Acad Sci U S A. 2017; 114(7):E1291-E1300. PMC: 5321001. DOI: 10.1073/pnas.1621150114. View

19.

Inoue F, Kreimer A, Ashuach T, Ahituv N, Yosef N . Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction. Cell Stem Cell. 2019; 25(5):713-727.e10. PMC: 6850896. DOI: 10.1016/j.stem.2019.09.010. View

20.

Long H, Prescott S, Wysocka J . Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution. Cell. 2016; 167(5):1170-1187. PMC: 5123704. DOI: 10.1016/j.cell.2016.09.018. View