Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer
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 acce...
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creator | Kim, Yuri Kim, Seong Won Saul, David Neyazi, Meraj Schmid, Manuel Wakimoto, Hiroko Slaven, Neil Lee, Joshua H Layton, Olivia G Wasson, Lauren K Letendre, Justin H Xiao, Feng Ewoldt, Jourdan K Gkatzis, Konstantinos Sommer, Peter Gobert, Bénédicte Wiest-Daesslé, Nicolas McAfee, Quentin Singhal, Nandita Lun, Mingyue Gorham, Joshua M Arany, Zoltan Sharma, Arun Toepfer, Christopher N Oudit, Gavin Y Pu, William T Dickel, Diane E Pennacchio, Len A Visel, Axel Chen, Christopher S Seidman, J G Seidman, Christine E |
description | 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 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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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 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.</description><identifier>EISSN: 1558-8238</identifier><identifier>PMID: 39688912</identifier><language>eng</language><publisher>United States</publisher><ispartof>The Journal of clinical investigation, 2024-12</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39688912$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, Yuri</creatorcontrib><creatorcontrib>Kim, Seong Won</creatorcontrib><creatorcontrib>Saul, David</creatorcontrib><creatorcontrib>Neyazi, Meraj</creatorcontrib><creatorcontrib>Schmid, Manuel</creatorcontrib><creatorcontrib>Wakimoto, Hiroko</creatorcontrib><creatorcontrib>Slaven, Neil</creatorcontrib><creatorcontrib>Lee, Joshua H</creatorcontrib><creatorcontrib>Layton, Olivia G</creatorcontrib><creatorcontrib>Wasson, Lauren K</creatorcontrib><creatorcontrib>Letendre, Justin H</creatorcontrib><creatorcontrib>Xiao, Feng</creatorcontrib><creatorcontrib>Ewoldt, Jourdan K</creatorcontrib><creatorcontrib>Gkatzis, Konstantinos</creatorcontrib><creatorcontrib>Sommer, Peter</creatorcontrib><creatorcontrib>Gobert, Bénédicte</creatorcontrib><creatorcontrib>Wiest-Daesslé, Nicolas</creatorcontrib><creatorcontrib>McAfee, Quentin</creatorcontrib><creatorcontrib>Singhal, Nandita</creatorcontrib><creatorcontrib>Lun, Mingyue</creatorcontrib><creatorcontrib>Gorham, Joshua M</creatorcontrib><creatorcontrib>Arany, Zoltan</creatorcontrib><creatorcontrib>Sharma, Arun</creatorcontrib><creatorcontrib>Toepfer, Christopher N</creatorcontrib><creatorcontrib>Oudit, Gavin Y</creatorcontrib><creatorcontrib>Pu, William T</creatorcontrib><creatorcontrib>Dickel, Diane E</creatorcontrib><creatorcontrib>Pennacchio, Len A</creatorcontrib><creatorcontrib>Visel, Axel</creatorcontrib><creatorcontrib>Chen, Christopher S</creatorcontrib><creatorcontrib>Seidman, J G</creatorcontrib><creatorcontrib>Seidman, Christine E</creatorcontrib><title>Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer</title><title>The Journal of clinical investigation</title><addtitle>J Clin Invest</addtitle><description>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 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.</description><issn>1558-8238</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><recordid>eNqFjskKwjAURYMgtg6_IO8HCh0lXYviWtxLbF9MpE3qS7Lo30sd1q4O53IWd8birKp4wvOCR2zp3CNNs7KsygWLinrHeZ3lMZNnvIdOeG0NWAlOUGN7JARpqf_MwrQgg2neog14haBQdF6NE8kD4TNoQgcCvPZ6qjxZoxtAo4RpkNZsLkXncPPlim2Ph8v-lAzh1mN7HUj3gsbr71jxN3gBTZJFYA</recordid><startdate>20241217</startdate><enddate>20241217</enddate><creator>Kim, Yuri</creator><creator>Kim, Seong Won</creator><creator>Saul, David</creator><creator>Neyazi, Meraj</creator><creator>Schmid, Manuel</creator><creator>Wakimoto, Hiroko</creator><creator>Slaven, Neil</creator><creator>Lee, Joshua H</creator><creator>Layton, Olivia G</creator><creator>Wasson, Lauren K</creator><creator>Letendre, Justin H</creator><creator>Xiao, Feng</creator><creator>Ewoldt, Jourdan K</creator><creator>Gkatzis, Konstantinos</creator><creator>Sommer, Peter</creator><creator>Gobert, Bénédicte</creator><creator>Wiest-Daesslé, Nicolas</creator><creator>McAfee, Quentin</creator><creator>Singhal, Nandita</creator><creator>Lun, Mingyue</creator><creator>Gorham, Joshua M</creator><creator>Arany, Zoltan</creator><creator>Sharma, Arun</creator><creator>Toepfer, Christopher N</creator><creator>Oudit, Gavin Y</creator><creator>Pu, William T</creator><creator>Dickel, Diane E</creator><creator>Pennacchio, Len A</creator><creator>Visel, Axel</creator><creator>Chen, Christopher S</creator><creator>Seidman, J G</creator><creator>Seidman, Christine E</creator><scope>NPM</scope></search><sort><creationdate>20241217</creationdate><title>Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer</title><author>Kim, Yuri ; 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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 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.</abstract><cop>United States</cop><pmid>39688912</pmid></addata></record> |
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title | Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer |
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