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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 27, 2024 |
| Title |
Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer [scRNA-Seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive the expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5 and MEF2 binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2 binding motif that was identified in a patient with unexplained DCM showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.
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| Overall design |
WT and mutant hiPSCs with TTN enhancer mutations were differentiated to cardiomyocytes. Cardiomyocytes from multiple independent differentiation were frozen. CellPlex kit was used to barcode each replicate and labeled cells were pooled together. Cells were sequenced using the standard single nuclei RNAseq protocol. TTN expression was compared between the WT hiPSC-CMs anad mutant hiPSC-CMs
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| Contributor(s) |
Kim S, Kim Y, M Gorham J |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Nov 23, 2024 |
| Last update date |
Nov 27, 2024 |
| Contact name |
Seong Won Kim |
| E-mail(s) |
[email protected]
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Street address |
77 Ave Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (8)
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| GSM8648866 |
Pooled snRNAseq of WT and MEF2_T>C CMs 1 |
| GSM8648867 |
Pooled snRNAseq of WT and MEF2_T>C CMs 2 |
| GSM8648868 |
Pooled snRNAseq of WT and E1 deletion CMs |
| GSM8648869 |
Pooled snRNAseq of WT and E0 deletion CMs |
| GSM8648870 |
Lipid Oligo Barcode library of WT and MEF2_T>C CMs 1 |
| GSM8648871 |
Lipid Oligo Barcode library of WT and MEF2_T>C CMs 2 |
| GSM8648872 |
Lipid Oligo Barcode library of WT and E1 deletion CMs |
| GSM8648873 |
Lipid Oligo Barcode library of WT and E0 deletion CMs |
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| Relations |
| BioProject |
PRJNA1189809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE282693_RAW.tar |
496.4 Mb |
(http)(custom) |
TAR (of MTX, TSV, TXT) |
SRA Run Selector |
| Raw data are available in SRA |
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