Functional analysis of human cardiac enhancers by tiling mutagenesis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE208284 | GEO | 2023/11/16
REPOSITORIES: GEO
Similar Datasets
Project description:Functional analysis of human cardiac enhancers by tiling mutagenesis
| PRJNA859125 | ENA
Project description:About 45% of congenital heart disease (CHD) is caused by rare gene mutations. Non-coding mutations that perturb cis-regulatory elements (CREs) likely contribute to CHD among the remaining cases without clear etiology. However, identifying CHD-causing non-coding variants has been problematic. We combined human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) differentiation and a lentivirus-mediated massively parallel reporter assay (lentiMPRA) to create a high-throughput platform to measure human cardiac enhancer activity. We tested 2451 candidate human cardiac enhancers, identified 1185 with measurable activity, and functionally dissected 123 of these by systematic tiling mutagenesis. We functionally evaluated 6761 non-coding de novo variants (ncDNVs) prioritized from the whole genome sequencing (WGS) of 749 CHD trios. 397 ncDNVs significantly affected cardiac CRE activity. Remarkably, 53% of these ncDNVs increased enhancer activity, often at regions with undetectable enhancer activity in the reference sequence. We introduced 10 of these DNVs associated with CHD genes into iPSCs and found that 4 altered expression of neighboring genes. Moreover, these 4 DNVs also altered cardiomyocyte differentiation, as assessed by single nucleus RNA sequencing. Using the MPRA data, we developed a regression model to prioritize future DNVs for functional testing and demonstrate that this model finds enrichment of DNVs in a second, independent WGS cohort. Taken together, we developed a scalable system to measure the impact of non-coding DNVs on CRE activity and deployed this platform to systematically assess the contribution of non-coding DNVs to CHD.
2022-08-05 | GSE210376 | GEO
Project description:We apply a massively parallel reporter assay (MPRA) that relies on mRNA and plasmid tag sequencing (Tag-Seq) to compare the regulatory activities of more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. The resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution and can be used the to train quantitative sequence-activity models (QSAMs). Reporter Tag-Seq from HEK293 cells transfected with each of six MPRA plasmid pools, with and without stimulation (forskolin or Sendai virus). The reporter mRNAs contain unique 10 nucleotide tags that facilitates quantitation of their abundances. The same tags were also sequenced from each ransfected plasmid pool to facilitate normalization to plasmid copy numbers. The reporter constructs were designed according to two different mutagenesis strategies: 'single-hit scanning' and 'multi-hit sampling'. The specific variants are included in the processed data files.
2012-02-26 | E-GEOD-31982 | biostudies-arrayexpress
Project description:To assess human cardiac enhancer activity, 400 bp candidate cardiac enhancers and controls were tested in iPSC-CMs using lentiMPRA.
2023-11-16 | GSE211417 | GEO
Project description:Analysis of the transcriptional changes in the heart resulting from the loss of cardiac enhancers. As there remains a limited understanding of the phenotypic consequences of enhancer mutations, we examined the impact of loss of function mutations by deleting two enhancers near heart disease genes in mice. In both cases, we observed loss of target gene expression, as well as cardiac phenotypes consistent with heart disease in humans, highlighting the functional importance of enhancers for normal heart function, as well as the potential contribution of enhancer mutations to heart disease. Hearts were dissected from wild-type and enhancer-null mice (either embryonic or adult) and processed for deep RNA-seq analysis.
2016-09-06 | E-GEOD-75907 | biostudies-arrayexpress
Project description:Functional dissection of human cardiac enhancers with hiPSC-CMs and lentiMPRA
| PRJNA870221 | ENA
Project description:We employ a massively parallel reporter assay (MPRA) to measure the ex vivo activities of hundreds of K562 and HepG2 enhancers with known transcription factor motif instances. For seven selected motifs that correspond to known or predicted activators and repressors in the two cell types, we make directed modifications of the bases corresponding to these motifs and observe the changes in enhancer activity. Reporter mRNA-seq from HepG2 and K562 cells transfected with a ~55,000-plex MPRA plasmid pool containing 5,418 mutated human enhancer sequences, each linked to 10 distinct 10-nt tags. The reporter mRNA tags facilitate quantitation of their abundances. The same tags were also sequenced from the transfected MPRA plasmid pool to facilitate normalization to plasmid copy numbers.
2013-03-15 | E-GEOD-33367 | biostudies-arrayexpress
Project description:We report the application of bioChIP-seq, bulk RNA-seq, Hi-C, and Massively parallel reporter assays (MPRAs) to characterize the p300-bound regulatory regions in murine cardiomyocytes (CMs). By obtaining ChIP-seq data of coactivator p300 from seven developmental stages of mouse CMs, we defined the dynamic p300 enhancers from embryonic CMs to adult CMs. We then validated the activity of dynamic p300 enhancers with AAV9-based MPRAs, we found dynamic p300 enhancers show dynamic activity from postnatal day 0 (P0) CMs to 4-week-old CMs. In addition, MRPA results suggest nuclear receptor motifs are required for the activity of some p300 late enhancers. With Hi-C data of E12.5, P0 and adult CMs, we identified chromatin structure changes such as chromatin loops, compartment switch, and new TAD boundaries are associated with dynamic p300 binding. This study provides data source of CM-selective p300 enhancers, chromatin 3D structure and gene expression during CM development and maturation.
2023-01-19 | GSE194087 | GEO
Project description:We report the application of bioChIP-seq, bulk RNA-seq, Hi-C, and Massively parallel reporter assays (MPRAs) to characterize the p300-bound regulatory regions in murine cardiomyocytes (CMs). By obtaining ChIP-seq data of coactivator p300 from seven developmental stages of mouse CMs, we defined the dynamic p300 enhancers from embryonic CMs to adult CMs. We then validated the activity of dynamic p300 enhancers with AAV9-based MPRAs, we found dynamic p300 enhancers show dynamic activity from postnatal day 0 (P0) CMs to 4-week-old CMs. In addition, MRPA results suggest nuclear receptor motifs are required for the activity of some p300 late enhancers. With Hi-C data of E12.5, P0 and adult CMs, we identified chromatin structure changes such as chromatin loops, compartment switch, and new TAD boundaries are associated with dynamic p300 binding. This study provides data source of CM-selective p300 enhancers, chromatin 3D structure and gene expression during CM development and maturation.
2023-01-19 | GSE195902 | GEO
Project description:We report the application of bioChIP-seq, bulk RNA-seq, Hi-C, and Massively parallel reporter assays (MPRAs) to characterize the p300-bound regulatory regions in murine cardiomyocytes (CMs). By obtaining ChIP-seq data of coactivator p300 from seven developmental stages of mouse CMs, we defined the dynamic p300 enhancers from embryonic CMs to adult CMs. We then validated the activity of dynamic p300 enhancers with AAV9-based MPRAs, we found dynamic p300 enhancers show dynamic activity from postnatal day 0 (P0) CMs to 4-week-old CMs. In addition, MRPA results suggest nuclear receptor motifs are required for the activity of some p300 late enhancers. With Hi-C data of E12.5, P0 and adult CMs, we identified chromatin structure changes such as chromatin loops, compartment switch, and new TAD boundaries are associated with dynamic p300 binding. This study provides data source of CM-selective p300 enhancers, chromatin 3D structure and gene expression during CM development and maturation.
2023-01-19 | GSE195901 | GEO