Browsing by Author "Pu, William T."

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    Author Correction: Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation
    (Springer Nature, 2021-08-19) VanDusen, Nathan J.; Lee, Julianna Y.; Gu, Weiliang; Butler, Catalina E.; Sethi, Isha; Zheng, Yanjiang; King, Justin S.; Zhou, Pingzhu; Suo, Shengbao; Guo, Yuxuan; Ma, Qing; Yuan, Guo-Cheng; Pu, William T.; Medical and Molecular Genetics, School of Medicine
    Correction to: Nature Communications 10.1038/s41467-021-24743-z, published online 21 July 2021. The original version of this Article contained an error in the spelling of the author William T. Pu, which was incorrectly given as William William Pu. This has now been corrected in both the PDF and HTML versions of the Article.
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    Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing
    (bioRxiv, 2024-12-04) Zheng, Yanjiang; Mayourian, Joshua; King, Justin S.; Li, Yifei; Bezzerides, Vassilios J.; Pu, William T.; VanDusen, Nathan J.; Pediatrics, School of Medicine
    The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.
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    Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation
    (Elsevier, 2023) Zhou, Pingzhu; VanDusen, Nathan J.; Zhang, Yanchun; Cao, Yangpo; Sethi, Isha; Hu, Rong; Zhang, Shuo; Wang, Guangyu; Ye, Lincai; Mazumdar, Neil; Chen, Jian; Zhang, Xiaoran; Guo, Yuxuan; Li, Bin; Ma, Qing; Lee, Julianna Y.; Gu, Weiliang; Gupta, Weiliang; Yuan, Guo-Cheng; Ren, Bing; Chen, Kaifu; Pu, William T.; Pediatrics, School of Medicine
    Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.
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    Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes
    (American Heart Association, 2022) Zheng, Yanjiang; VanDusen, Nathan J.; Butler, Catalina E.; Ma, Qing; King, Justin S.; Pu, William T.; Pediatrics, School of Medicine
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    In Vivo Dissection of Chamber-Selective Enhancers Reveals Estrogen-Related Receptor as a Regulator of Ventricular Cardiomyocyte Identity
    (Wolters Kluwer, 2023) Cao, Yangpo; Zhang, Xiaoran; Akerberg, Brynn N.; Yuan, Haiyun; Sakamoto, Tomoya; Xiao, Feng; VanDusen, Nathan J.; Zhou, Pingzhu; Sweat, Mason E.; Wang, Yi; Prondzynski, Maksymilian; Chen, Jian; Zhang, Yan; Wang, Peizhe; Kelly, Daniel P.; Pu, William T.; Pediatrics, School of Medicine
    Background: Cardiac chamber-selective transcriptional programs underpin the structural and functional differences between atrial and ventricular cardiomyocytes (aCMs and vCMs). The mechanisms responsible for these chamber-selective transcriptional programs remain largely undefined. Methods: We nominated candidate chamber-selective enhancers (CSEs) by determining the genome-wide occupancy of 7 key cardiac transcription factors (GATA4, MEF2A, MEF2C, NKX2-5, SRF, TBX5, TEAD1) and transcriptional coactivator P300 in atria and ventricles. Candidate enhancers were tested using an adeno-associated virus-mediated massively parallel reporter assay. Chromatin features of CSEs were evaluated by performing assay of transposase accessible chromatin sequencing and acetylation of histone H3 at lysine 27-HiChIP on aCMs and vCMs. CSE sequence requirements were determined by systematic tiling mutagenesis of 29 CSEs at 5 bp resolution. Estrogen-related receptor (ERR) function in cardiomyocytes was evaluated by Cre-loxP-mediated inactivation of ERRα and ERRγ in cardiomyocytes. Results: We identified 134 066 and 97 506 regions reproducibly occupied by at least 1 transcription factor or P300, in atria or ventricles, respectively. Enhancer activities of 2639 regions bound by transcription factors or P300 were tested in aCMs and vCMs by adeno-associated virus-mediated massively parallel reporter assay. This identified 1092 active enhancers in aCMs or vCMs. Several overlapped loci associated with cardiovascular disease through genome-wide association studies, and 229 exhibited chamber-selective activity in aCMs or vCMs. Many CSEs exhibited differential chromatin accessibility between aCMs and vCMs, and CSEs were enriched for aCM- or vCM-selective acetylation of histone H3 at lysine 27-anchored loops. Tiling mutagenesis of 29 CSEs identified the binding motif of ERRα/γ as important for ventricular enhancer activity. The requirement of ERRα/γ to activate ventricular CSEs and promote vCM identity was confirmed by loss of the vCM gene profile in ERRα/γ knockout vCMs. Conclusions: We identified 229 CSEs that could be useful research tools or direct therapeutic gene expression. We showed that chamber-selective multi-transcription factor, P300 occupancy, open chromatin, and chromatin looping are predictive features of CSEs. We found that ERRα/γ are essential for maintenance of ventricular identity. Finally, our gene expression, epigenetic, 3-dimensional genome, and enhancer activity atlas provide key resources for future studies of chamber-selective gene regulation.
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    Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation
    (Springer Nature, 2021-07-21) VanDusen, Nathan J.; Lee, Julianna Y.; Gu, Weiliang; Butler, Catalina E.; Sethi, Isha; Zheng, Yanjiang; King, Justin S.; Zhou, Pingzhu; Suo, Shengbao; Guo, Yuxuan; Ma, Qing; Yuan, Guo-Cheng; Pu, William T.; Medical and Molecular Genetics, School of Medicine
    The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.