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Predictable Engineering of Signal-Dependent Cis-Regulatory Elements

ABSTRACT: Cis-regulatory elements (CREs) control how genes respond to external signals, but the principles governing their structure and function remain poorly understood. While differential transcription factor binding is known to regulate gene expression, how CREs integrate the amount and combination of inputs to secure precise spatiotemporal profiles of gene expression remains unclear. Here, we developed a high-throughput combinatorial screening strategy, that we term NeMECiS , to investigate signal-dependent synthetic CREs (synCREs) in differentiating mammalian stem cells. By concatenating fragments of functional CREs from genes that respond to Sonic Hedgehog in the developing vertebrate neural tube, we found that CRE activity follows hierarchical design rules. While individual 200-base-pair fragments showed minimal activity, their combinations generated thousands of functional signal-responsive synCREs, many exceeding the activity of natural sequences. Statistical modelling revealed CRE function can be decomposed into specific quantitative contributions in which sequence fragments combine through a multiplicative rule, tuned by their relative positioning and spacing. These findings provide a predictive framework for CRE redesign, which we used to engineer synthetic CREs that alter the pattern of motor neuron differentiation in neural tissue. These findings establish quantitative principles for engineering synthetic regulatory elements with programmable signal responses to rewire genetic circuits and control stem cell differentiation, providing a basis for understanding developmental gene regulation and designing therapeutic gene expression systems.

ORGANISM(S): synthetic construct Mus musculus

PROVIDER: GSE291283 | GEO | 2025/03/07

REPOSITORIES: GEO

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Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC and pcHiC data and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: Promotor capture libraries were constructed via the following method. Crosslinked cells were lysed, digested with DpnII, ends filled in using biotin-14-dATP followed by overnight proximity ligation. Ligated samples underwent crosslink reversal, DNA extraction with proteinase K, and biotin removal from unligated fragments. DNA was fragmented to 500-600 bp, column purified, and biotinylated fragments pulled down. Biotinylated fragments were converted into Illumina compatible libraries using the SureSelect XT HS2 DNA System and a custom SureSelect probe set (design ID S3346176) (Agilent Technologies) according to the manufacture’s protocol with the following exception: pre-capture PCR was performed as four 50 ul reactions that were recombined during clean-up. The quality and concentration of the libraries were assessed using the High Sensitivity D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions. Libraries were sequenced 150 bp paired end on an Illumina NovaSeq 6000.

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Predictable Engineering of Signal-Dependent Cis-Regulatory Elements

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