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Predictable Engineering of Signal-Dependent Cis-Regulatory Elements

ABSTRACT: Predictable Engineering of Signal-Dependent Cis-Regulatory Elements

PROVIDER: PRJNA1232713 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32596800_1.fastq.gz	Fastqsanger.gz
	SRR32596800_2.fastq.gz	Fastqsanger.gz
	SRR32596801_1.fastq.gz	Fastqsanger.gz
	SRR32596801_2.fastq.gz	Fastqsanger.gz
	SRR32596802_1.fastq.gz	Fastqsanger.gz

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Predictable Engineering of Signal-Dependent Cis-Regulatory Elements

Project description: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.

2025-03-07 | GSE291283 | GEO

Effects of cis-regulatory elements on proteome

Project description:Background Genomic variations contribute to the phenotypic diversity of individuals. A number of polymorphisms in protein-coding regions that alter drug efficacy or lead to adverse reactions have been characterized; however, noncoding regions that affect drug responses are largely overlooked, except for a limited number of well-studied enhancers. Results We conducted a quantitative assessment of cis-regulatory elements (CREs) based on transcription initiation profiling of mRNAs and noncoding RNAs, including enhancer RNAs, by using CAGE (Cap Analysis of Gene Expression). Candidate CREs identified in a hepatocellular carcinoma HepG2 cell line with stable expression of drug-responsive transcription factor pregnane X receptor (PXR) were further narrowed down by integrating data of PXR-binding sites in human primary hepatocytes and genome-wide association studies. We found more than 100-fold enrichments of the candidates to genetically associated loci with circulating levels of bilirubin and vitamin D, which implicated a link to adverse reactions of PXR ligands. We uncovered novel enhancers of UGT1A1 and TSKU through CRISPR/Cas9 knockout experiments. We identified alleles altering regulatory activities of UGT1A1 and CYP24A1enhancers by using luciferase reporter assay. Furthermore, our siRNA experiments revealed an unexpected impact of TSKU on the expression of vitamin D-metabolizing enzymes. Conclusions Our transcriptome-based assessment of CREs expanded the list of drug-inducible and PXR-mediated enhancers and super-enhancers. We identified regulatory alleles that alter drug-induced gene expressions, and discovered a novel molecular cascade associated with an adverse reaction. Our results contribute a precise understanding of the noncoding elements of the human genome underlying drug responses.

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Project description:The goal of this study is to improve the quality, efficiency, and sustainability of milk production by improving the understanding of the function of cis-regulatory elements in regulating the bovine mammary gland. To this end, we have characterized the non-lactating and lactating mammary gland transcriptomes by whole transcriptome shotgun sequencing (RNA-seq). We will identify cis-regulatory elements in the non-lactating and lactating bovine mammary gland genome-wide. Finally, we will annotate and characterize mammary gland cis-regulatory elements by computational analysis and identify high-resolution genome-wide in vivo footprints of diverse trans-acting-factors (TF), over-represented TF bindings sites and overlapping SNPs.

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