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While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960 , a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features., Competing Interests: JM, NA, HM, KH, MB, SW, JS, SW, TS, PC, LS No competing interests declared, JW J.W. is a paid member of Camp4 and Paratus biosciences scientific advisory boards, (© 2024, Mohammed et al.)

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution., Competing Interests: Declaration of interests J.W. is a paid scientific advisory board member at Camp4 and Paratus Sciences. J.T. has a consultancy agreement with DeepMind Technologies. J.W. is an advisory board member at Cell Press journals, including Cell, Molecular Cell, and Developmental Cell., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)