Robert Frömel, Julia Rühle, Aina Bernal Martinez, Chelsea Szu-Tu, Felix Pacheco Pastor et al.
During cellular differentiation, enhancers transform overlapping gradients of transcription factors (TFs) to highly specific gene expression patterns. However, the vast complexity of regulatory DNA impedes the identification of the underlying cis-regulatory rules. Here, we characterized 64,400 fully synthetic DNA sequences to bottom-up dissect design principles of cell-state-specific enhancers in the context of the differentiation of blood stem cells to seven myeloid lineages. Focusing on binding sites for 38 TFs and their pairwise interactions, we found that identical sites displayed both repressive and activating function as a consequence of cell state, site combinatorics, or simply predicted occupancy of a TF on an enhancer. Surprisingly, combinations of activating sites frequently neutralized one another or gained repressive function. These negative synergies convert quantitative imbalances in TF expression into binary activity patterns. We exploit this principle to automatically create enhancers with specificity to user-defined combinations of hematopoietic progenitor cell states from scratch.
Robert Frömel, Julia Rühle, Aina Bernal Martinez, Chelsea Szu-Tu, Felix Pacheco Pastor, Rosa Martinez-Corral, Lars Velten. Design principles of cell-state-specific enhancers in hematopoiesis. 2025-05-08. https://doi.org/10.1016/j.cell.2025.04.017
Library G tested 1,667 genomic elements selected from differentially accessible regions and enhancer/promoter annotations in the primary murine HSPC culture system. The genomic library was measured across six available differentiated progenitor states.
The Library B pairwise synthetic enhancer library was delivered to human K-562 cells using a minimal CMV promoter rather than the standard minimal promoter. The duplicate screen tests promoter-context robustness of the same pairwise design space.
The Library A single-factor synthetic enhancer library was delivered by lentivirus to human K-562 cells and measured in duplicate at the early post-transduction time point. The table contains one K-562 measurement per construct with motif-design annotations.
This is the public archive's across-cell-state aggregate DATA object for Library C, providing a single aggregate activity view for the pairwise synthetic enhancer library. It is a derived analysis view rather than an additional biological infection.
The broad Library C pairwise motif library covering 42 transcription factors was delivered to human K-562 cells using the standard minimal promoter and measured in duplicate at the early post-transduction time point.
Library C sampled a broad space of pairwise combinations covering 42 transcription factors, including different motif affinities, orientations, spacings, and arrangements in random DNA. The library was measured in seven differentiated murine HSPC progenitor states.
Library H tested complex synthetic enhancers containing three to twelve randomly combined high-affinity motifs from the selected transcription factors, with motif identity, orientation, and spacing varied in random DNA. The library was measured across seven differentiated murine HSPC progenitor states.
Library D tested model-designed synthetic enhancers intended to produce user-specified combinations of activation, inactivity, or repression across six hematopoietic cell states. The designs were initialized randomly or with a random-forest model and optimized by local or global sequence search before measurement in primary murine HSPC differentiation cultures.
Library F tested synthetic combinations centered on Fli1–Spi1 and Gata2–Cebpa, retaining motif counts and affinity sums for the four factors. The library was measured across six available differentiated murine HSPC progenitor states.
The Library B pairwise synthetic enhancer library was delivered to human K-562 cells using the standard minimal promoter and measured at the later post-infection time point to test temporal stability of activity.
Library A tested one to six motifs for each of 38 transcription factors, varying motif number, affinity, orientation, arrangement, and spacing in random DNA. The lentiviral library was measured across seven FACS-defined progenitor states generated from differentiating primary murine HSPCs.
Library B tested systematic pairwise combinations among a core set of ten hematopoietic transcription factors, varying pair affinity, orientation, spacing, site number, and arrangement in random DNA. The lentiviral library was measured across seven differentiated murine HSPC progenitor states.
The systematic Library B pairwise motif library was delivered to human K-562 cells using the standard minimal promoter and measured at the early post-transduction time point in duplicate. Pair identity, affinity, spacing, orientation, and arrangement annotations are retained.
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