Drew T. Bergman, Thouis R. Jones, Vincent Liu, Judhajeet Ray, Evelyn Jagoda et al.
Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters. A proposed model for this specificity is that promoters have sequence-encoded preferences for certain enhancers, for example, mediated by interacting sets of transcription factors or cofactors. This ‘biochemical compatibility’ model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila. However, the degree to which human enhancers and promoters are intrinsically compatible has not yet been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we design a high-throughput reporter assay called enhancer × promoter self-transcribing active regulatory region sequencing (ExP STARR-seq) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer–promoter compatibility, whereby most enhancers activate all promoters by similar amounts, and intrinsic enhancer and promoter activities multiplicatively combine to determine RNA output (R² = 0.82). In addition, two classes of enhancers and promoters show subtle preferential effects. Promoters of housekeeping genes contain built-in activating motifs for factors such as GABPA and YY1, which decrease the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lack these motifs and show stronger responsiveness to enhancers. Together, this systematic assessment of enhancer–promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.
Drew T. Bergman, Thouis R. Jones, Vincent Liu, Judhajeet Ray, Evelyn Jagoda, Layla Siraj, Helen Y. Kang, Joseph Nasser, Michael Kane, Antonio Rios, Tung H. Nguyen, Sharon R. Grossman, Charles P. Fulco, Eric S. Lander, Jesse M. Engreitz. Compatibility rules of human enhancer and promoter sequences. 2022-05-20. https://doi.org/10.1038/s41586-022-04877-w
Tagmented K562 genomic DNA was hybrid-selected against probes targeting accessible genomic elements, producing 300–700-bp fragments with 10-bp UMIs for a targeted STARR-seq library. Four K562 biological replicates were sequenced to quantify RNA/DNA activity for millions of accessible-element fragments.
A focused ExP STARR-seq library tested natural promoter and enhancer sequences together with promoter motif insertions, motif disruptions, and synthetic motif arrays. Edited and unedited constructs were measured in four K562 reporter-RNA replicates and two plasmid-DNA input replicates.
A plasmid library paired approximately 1,000 synthetic 264-bp human enhancer sequences with approximately 1,000 264-bp promoter sequences in all combinations, assigning a unique 16-bp plasmid barcode to each construct. The library was transiently transfected into K562 cells in four biological replicates, and reporter RNA was quantified relative to plasmid DNA input.
Tagmented K562 genomic DNA was hybrid-selected against probes targeting gene promoters, producing 300–700-bp fragments with 10-bp UMIs for a targeted STARR-seq library. Four K562 biological replicates were sequenced to quantify RNA/DNA activity for millions of promoter-region fragments.
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 7 files (ZIP)GSE184426_ExP_1Kx1K_counts.txt.gzGSE184426_family.soft.gzGSE184426_HS-STARR-seq-enhancers_counts.txt.gzGSE184426_HS-STARR-seq-promoters_counts.txt.gzGSE184426_starr_seq_100x100_all_experimental_counts_file.txt.gzSupplementary_Table_10_Sequences.xlsxSupplementary_Table_3_Luciferase.xlsx