Study / S0TWAP8592020-06-29

Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays

Jessica E. Davis, Kimberly D. Insigne, Eric M. Jones, Quinn A. Hastings, W. Clifford Boldridge et al.

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About this study

In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.

Full author list & citation

Jessica E. Davis, Kimberly D. Insigne, Eric M. Jones, Quinn A. Hastings, W. Clifford Boldridge, Sriram Kosuri. Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays. 2020-06-29. https://doi.org/10.1016/j.cels.2020.05.011

Experiments 5

E0K9GDHVO

Targeted genomic-integration MPRA at the H11 landing pad

The SP3/SP5 library was integrated into the defined H11 landing pad in HEK293T cells by BxB1 recombination and measured at 8 µM forskolin in two biological replicates, each with two DNA technical replicates.

Targeted Genomic Integration MPRAHumanGRCh38

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E4N6QMVOZ

Dose-response episomal MPRA: 0–64 µM forskolin

SP3 and SP5 synthetic CRE libraries were assayed episomally across nine forskolin concentrations, with two biological replicates per condition.

Episomal Plasmid MPRAHumanGRCh38

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E4O6HQ2AE

Follow-up episomal MPRA: CRE backgrounds and spacing at 0 versus 4 µM forskolin

The follow-up SP5-derived library tests six-site CRE configurations, two-site spacing/distance variants, and 10-bp background scrambles in an episomal MPRA with two biological replicates at baseline and 4 µM forskolin.

Episomal Plasmid MPRAHumanGRCh38

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E4T22QLCR

Main episomal MPRA: logarithmic forskolin dose series

The main SP3/SP5 episomal CRE library was measured in two biological replicates over a logarithmic forskolin series from baseline through 4 µM.

Episomal Plasmid MPRAHumanGRCh38

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E7SQ3WH68

Initial episomal MPRA: 0 versus 25 µM forskolin

Broad episomal library containing the CRE spacing/distance, CRE number/affinity, single-CRE designs, and expression controls, measured in two biological replicates at baseline and 25 µM forskolin.

Episomal Plasmid MPRAHumanGRCh38

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Raw source data 57 files

Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.

Download all 57 files (ZIP)GSE137922_barcode_statistics_followup.txt.gz GSE137922_family.soft.gz GSE137922_uniqueSP2345.txt.gz GSM4094373_20170320_bc_DNA.txt.gz GSM4094374_20170320_bc_R0A.txt.gz GSM4094375_20170320_bc_R0B.txt.gz GSM4094376_20170320_bc_R25A.txt.gz GSM4094377_20170320_bc_R25B.txt.gz GSM4094378_20170631_DNA_BC.txt.gz GSM4094379_20170631_R0A_BC.txt.gz GSM4094380_20170631_R0B_BC.txt.gz GSM4094381_20170631_R16A_BC.txt.gz GSM4094382_20170631_R16B_BC.txt.gz GSM4094383_20170631_R1A_BC.txt.gz GSM4094384_20170631_R1B_BC.txt.gz GSM4094385_20170631_R25A_BC.txt.gz GSM4094386_20170631_R25B_BC.txt.gz GSM4094387_20170631_R2A_BC.txt.gz GSM4094388_20170631_R2B_BC.txt.gz GSM4094389_20170631_R32A_BC.txt.gz GSM4094390_20170631_R32B_BC.txt.gz GSM4094391_20170631_R4A_BC.txt.gz GSM4094392_20170631_R4B_BC.txt.gz GSM4094393_20170631_R64A_BC.txt.gz GSM4094394_20170631_R64B_BC.txt.gz GSM4094395_20170631_R8A_BC.txt.gz GSM4094396_20170631_R8B_BC.txt.gz GSM4094397_20170921_DNA_BC.txt.gz GSM4094398_20170921_R0A_BC.txt.gz GSM4094399_20170921_R0B_BC.txt.gz GSM4094400_20170921_R20A_BC.txt.gz GSM4094401_20170921_R20B_BC.txt.gz GSM4094402_20170921_R2-1A_BC.txt.gz GSM4094403_20170921_R2-1B_BC.txt.gz GSM4094404_20170921_R2-2A_BC.txt.gz GSM4094405_20170921_R22A_BC.txt.gz GSM4094406_20170921_R2-2B_BC.txt.gz GSM4094407_20170921_R22B_BC.txt.gz GSM4094408_20170921_R2-3A_BC.txt.gz GSM4094409_20170921_R2-3B_BC.txt.gz GSM4094410_20170921_R2-4A_BC.txt.gz GSM4094411_20170921_R2-4B_BC.txt.gz GSM4094412_20170921_R2-5A_BC.txt.gz GSM4094413_20170921_R2-5B_BC.txt.gz GSM4094414_20171129_DNA_1_1_BC.txt.gz GSM4094415_20171129_DNA_1_2_BC.txt.gz GSM4094416_20171129_DNA_2_1_BC.txt.gz GSM4094417_20171129_DNA_2_2_BC.txt.gz GSM4094418_20171129_R8_1_BC.txt.gz GSM4094419_20171129_R8_2_BC.txt.gz GSM4094420_20190712_DNA_BC.txt.gz GSM4094421_20190712_R0A_BC.txt.gz GSM4094422_20190712_R0B_BC.txt.gz GSM4094423_20190712_R4A_BC.txt.gz GSM4094424_20190712_R4B_BC.txt.gz Supplemental_Data.pdf Supplemental_Information.pdf