Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis.

ABSTRACT: We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biology.

SUBMITTER: Patwardhan RP

PROVIDER: S-EPMC2849652 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis.

Patwardhan Rupali P RP Lee Choli C Litvin Oren O Young David L DL Pe'er Dana D Shendure Jay J

Nature biotechnology 20091201 12

We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biolo ...[more]

PMID: 19915551

Similar Datasets

Saturation mutagenesis of twenty disease-associated regulatory elements at single base-pair resolution.

Project description:The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

| S-EPMC6687891 | biostudies-literature

Saturation mutagenesis of disease-associated regulatory elements

Project description:The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we performed saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitution and deletion mutations. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or various integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and also comprise a gold-standard dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

2019-02-15 | GSE126550 | GEO

Saturation mutagenesis of disease-associated regulatory elements

Project description:Saturation mutagenesis of disease-associated regulatory elements

| PRJNA522353 | ENA

Coupling high-throughput mapping with proteomics analysis delineates cis-regulatory elements at high resolution.

Project description:Growing evidence suggests that functional cis-regulatory elements (cis-REs) not only exist in epigenetically marked but also in unmarked sites of the human genome. While it is already difficult to identify cis-REs in the epigenetically marked sites, interrogating cis-REs residing within the unmarked sites is even more challenging. Here, we report adapting Reel-seq, an in vitro high-throughput (HTP) technique, to fine-map cis-REs at high resolution over a large region of the human genome in a systematic and continuous manner. Using Reel-seq, as a proof-of-principle, we identified 408 candidate cis-REs by mapping a 58 kb core region on the aging-related CDKN2A/B locus that harbors p16INK4a. By coupling Reel-seq with FREP-MS, a proteomics analysis technique, we characterized two cis-REs, one in an epigenetically marked site and the other in an epigenetically unmarked site. These elements are shown to regulate the p16INK4a expression over an ∼100 kb distance by recruiting the poly(A) binding protein PABPC1 and the transcription factor FOXC2. Downregulation of either PABPC1 or FOXC2 in human endothelial cells (ECs) can induce the p16INK4a-dependent cellular senescence. Thus, we confirmed the utility of Reel-seq and FREP-MS analyses for the systematic identification of cis-REs at high resolution over a large region of the human genome.

| S-EPMC8754656 | biostudies-literature

Economical analysis of saturation mutagenesis experiments.

Project description:Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

| S-EPMC4507136 | biostudies-other

Genome-wide High Resolution Mapping of Active Regulatory DNA Elements in the Rice Genome

Project description:This SuperSeries is composed of the following subset Series: GSE26610: DNase I hypersensitive sites in two tissues of rice GSE26733: ChIP-seq to identify the positions of three histone modifications in the rice genome Refer to individual Series

2011-11-09 | E-GEOD-26734 | biostudies-arrayexpress

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis.

Project description:Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5' upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

| S-EPMC4446435 | biostudies-literature

Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library.

Project description:BackgroundThe identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified.ResultsHere we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli.ConclusionsSuccessful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

| S-EPMC3923269 | biostudies-literature

Genome-wide High Resolution Mapping of Active Regulatory DNA Elements in the Rice Genome

Project description:This SuperSeries is composed of the SubSeries listed below.

2011-11-09 | GSE26734 | GEO

DNA-Diffusion: Leveraging Generative Models for Controlling Chromatin Accessibility and Gene Expression via Synthetic Regulatory Elements.

Project description:The challenge of systematically modifying and optimizing regulatory elements for precise gene expression control is central to modern genomics and synthetic biology. Advancements in generative AI have paved the way for designing synthetic sequences with the aim of safely and accurately modulating gene expression. We leverage diffusion models to design context-specific DNA regulatory sequences, which hold significant potential toward enabling novel therapeutic applications requiring precise modulation of gene expression. Our framework uses a cell type-specific diffusion model to generate synthetic 200 bp regulatory elements based on chromatin accessibility across different cell types. We evaluate the generated sequences based on key metrics to ensure they retain properties of endogenous sequences: transcription factor binding site composition, potential for cell type-specific chromatin accessibility, and capacity for sequences generated by DNA diffusion to activate gene expression in different cell contexts using state-of-the-art prediction models. Our results demonstrate the ability to robustly generate DNA sequences with cell type-specific regulatory potential. DNA-Diffusion paves the way for revolutionizing a regulatory modulation approach to mammalian synthetic biology and precision gene therapy.

| S-EPMC10862870 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data