Similar Datasets
Project description:Dense, systematic mutagenesis to identify the features required for CSEs
| PRJNA888108 | ENA
Project description:To further determine the sequence features of active cardiac enhancers, 123 cardiac enhancers were functionally dissected by 10 bp tiling deletion-based mutagenesis MPRA in iPSC-CMs.
2023-11-16 | GSE208284 | GEO
Project description:We used CRISPR KI technology to mutagenize the cis-regulatory elements including the editing stem and the editing complementary sequence of three RNA editing substrates-NEIL1, TTYH2, AJUBA. We designed single nucleotide variants, double-nucleotide variants and other mutant isoforms to interrogate the primary sequence and the secondary structures around the endogenous RNA editing sites. Gene specific primers were used to make the NGS library for each locus. The unique mutation of each tested isoform can be used as barcode and RNA editing level can be measured for each isoform from the pooled library. We found that RNA sequence and structure features synergistically determine the editing levels. Several features, such as mutation number, free energy, and probability of active conformation, play an important role in determining editing efficiency. This study systematically investigated the contribution of cis-regulatory elements to ADAR1 RNA editing by combining CRISPR-based saturation mutagenesis and deep sequencing technology.
2020-06-01 | GSE138860 | GEO
Project description:Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation.
2023-11-09 | GSE247431 | GEO
Project description:Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation.
2023-11-09 | GSE247414 | GEO
Project description:Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation.
2023-11-09 | GSE247412 | GEO
Project description:Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation.
2023-11-09 | GSE247411 | GEO
Project description:This RNA sequencing aimed to verify mito-ncR-805 upregulation in CSE-treated cells, and determine its exact sequence in alveolar type-II cells.
2020-08-30 | GSE131439 | GEO
Project description:Neurovascular dysfunction in penis is a fundamental reason of erectile dysfunction. Diabetes mellitus is one of the major causes of erectile dysfunction and leads to a poor response to oral phosphodiesterase-5 inhibitors. Heat shock protein 70 (Hsp70), a ubiquitous molecular chaperone exist in all living organisms, is known to play a role in cell survival and neuroprotection. Here, we report an effectiveness of Hsp70 in mediating neurovascular regeneration in diabetic conditions. Using Hsp70-Tg mice or Hsp70 protein administration, we demonstrate that overexpression of Hsp70 in diabetic mice restores erectile function through enhanced penile angiogenesis and neural regeneration. We found that cystathionine gamma-lyase (Cse) is a novel target of Hsp70-driven penile angiogenesis and neural regeneration. Hsp70-Cse triggered SDF1/HO-1/PI3K/Akt/eNOS/NF-κB p65 pathways involved in angiogenesis and neural regeneration. Coimmunoprecipitation and His-Tag pull down assay using mouse cavernous endothelial cells treated with Hsp70 showed the physical interaction between Hsp70 and Cse, and solid-phase binding assay revealed a high-affinity Hsp70-Cse binding with an apparent dissociation constant of 1.8 nmol/L. We provide a novel and solid evidence for Cse-dependent mechanism of Hsp70, which mediates Hsp70-induced neurovascular regeneration and the restoration of erectile function under diabetic conditions.
2022-05-11 | GSE180916 | GEO
Project description:Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs. Despite a compelling need towards predictive understanding of natural and engineered editing events, how the RNA sequence and structure determine the editing efficiency and specificity (i.e., cis-regulation) is poorly understood. We apply a CRISPR/Cas9-mediated saturation mutagenesis approach to generate libraries of mutations near three natural editing substrates at their endogenous genomic loci. We use machine learning to integrate diverse RNA sequence and structure features to model editing levels measured by deep sequencing. We confirm known features and identify new features important for RNA editing. Training and testing XGBoost algorithm within the same substrate yield models that explain 68 to 86 percent of substrate-specific variation in editing levels. However, the models do not generalize across substrates, suggesting complex and context-dependent regulation patterns. Our integrative approach can be applied to larger scale experiments towards deciphering the RNA editing code.
2021-03-05 | GSE168234 | GEO