Dana M. King, Clarice Kit Yee Hong, James L. Shepherdson, David M. Granas, Brett B. Maricque et al.
In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.
Dana M. King, Clarice Kit Yee Hong, James L. Shepherdson, David M. Granas, Brett B. Maricque, Barak A. Cohen. Synthetic and genomic regulatory elements reveal aspects of cis-regulatory grammar in mouse embryonic stem cells. 2020-02-11. https://doi.org/10.7554/eLife.41279
A follow-up plasmid MPRA tested 30 constructs formed from six synthetic 4-mer TF-binding-site arrangements and five spacer variants, including the original spacer. Each construct was designed with five barcodes and was mixed with a small portion of the SYN library.
A plasmid MPRA tested 624 synthetic 2-mer, 3-mer, and 4-mer elements containing consensus SOX2, OCT4/POU5F1, KLF4, and ESRRB binding sites in all allowed orders and orientations. Each primary element was represented by eight unique barcodes and compared with basal-promoter controls.
A plasmid MPRA tested mouse genomic 81–82 bp elements selected for comparable pluripotency-factor binding-site configurations alongside matched sequences in which the primary sites were disrupted. The processed table contains one row per recovered gWT/gMUT coordinate pair.
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 13 files (ZIP)elife-41279-supp1-v2.xlsxGSE120240_family.soft.gzGSE120240_RAW.tarGSM3396576_SYN_Rep1_mRNA.counts.txt.gzGSM3396577_SYN_Rep2_mRNA.counts.txt.gzGSM3396578_SYN_Rep3_mRNA.counts.txt.gzGSM3396579_SYN_plasmid_DNA.counts.txt.gzGSM3396580_GEN_Rep1_mRNA.counts.txt.gzGSM3396581_GEN_Rep2_mRNA.counts.txt.gzGSM3396582_GEN_Rep3_mRNA.counts.txt.gzGSM3396583_GEN_plasmid_DNA.counts.txt.gzking41279_article.pdfsource_notes.txt