Browsing by Author "Kim, Il-Man"

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    Decoding the Gene Regulatory Network of Muscle Stem Cells in Mouse Duchenne Muscular Dystrophy: Revelations from Single-Nuclei RNA Sequencing Analysis
    (MDPI, 2023-08-05) Shen, Yan; Kim, Il-Man; Tang, Yaoliang; Anatomy, Cell Biology and Physiology, School of Medicine
    The gene dystrophin is responsible for Duchenne muscular dystrophy (DMD), a grave X-linked recessive ailment that results in respiratory and cardiac failure. As the expression of dystrophin in muscle stem cells (MuSCs) is a topic of debate, there exists a limited understanding of its influence on the gene network of MuSCs. This study was conducted with the objective of investigating the effects of dystrophin on the regulatory network of genes in MuSCs. To comprehend the function of dystrophin in MuSCs from DMD, this investigation employed single-nuclei RNA sequencing (snRNA-seq) to appraise the transcriptomic profile of MuSCs obtained from the skeletal muscles of dystrophin mutant mice (DMDmut) and wild-type control mice. The study revealed that the dystrophin mutation caused the disruption of several long non-coding RNAs (lncRNAs), leading to the inhibition of MEG3 and NEAT1 and the upregulation of GM48099, GM19951, and GM15564. The Gene Ontology (GO) enrichment analysis of biological processes (BP) indicated that the dystrophin mutation activated the cell adhesion pathway in MuSCs, inhibited the circulatory system process, and affected the regulation of binding. The study also revealed that the metabolic pathway activity of MuSCs was altered. The metabolic activities of oxidative phosphorylation (OXPHOS) and glycolysis were elevated in MuSCs from DMDmut. In summary, this research offers novel insights into the disrupted gene regulatory program in MuSCs due to dystrophin mutation at the single-cell level.
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    Identifying Novel Causes of X-Linked Heterotaxy
    (2024-05) Wells, John Robert; Ware, Stephanie M.; Firulli, Anthony B.; Kim, Il-Man; Landis, Benjamin J.
    Heterotaxy is a congenital disorder characterized by abnormal arrangement of formation of thoracic and abdominal organs due to errors in embryonic left-right patterning, affecting ~1 in 10,000 live births. These patients exhibit considerable phenotypic heterogeneity, with structural heart defects significantly contributing to poor outcomes. Variants in several genes can disrupt laterality, but ZIC3 variants, primarily identified through targeted sequencing of its coding region, are the only recognized cause of X-liked heterotaxy. This dissertation focuses on a heterotaxy pedigree with four affected males, demonstrating an X-linked inheritance. No coding variant in ZIC3 was identified, leaving the pedigree unsolved for over two decades. Initially, the family’s heterotaxy was hypothesized to be caused by a coding variant in a novel heterotaxy locus on the X chromosome. X-exome sequencing identified a missense variant in GPR101, a gene whose closest phylogenetic relative has been implicated in left-right asymmetry in zebrafish. However, subsequent findings from this study and other research groups suggests GPR101 does not regulate left-right patterning, making the hypothesized GPR101 variant unlikely to be disease-causative. The next hypothesis explored was a non-coding variant in ZIC3, undetected by X-exome sequencing. Whole genome sequencing identified a novel, deep intronic variant in ZIC3, initially hypothesized to trigger the inclusion of an intronic sequence as a pseudoexon during RNA splicing. Further analysis revealed the variant profoundly altered RNA splicing, resulting in the production of several novel ZIC3 isoforms and reduced expression of normal ZIC3 protein. These novel isoforms displayed abnormal function in a variety of in vitro and in vivo assays. This marks the first reported instance of pseudoexon inclusion associated with heterotaxy for any gene and underscores the critical need to expand the scope of variant evaluation beyond mere missense and nonsense variants. The clinical and research field must adapt to assess non-coding variants and to consider alternative disease mechanisms, such as abnormal splicing or dysregulated expression of key left-right patterning genes, in unresolved heterotaxy cases.
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    LncRNA HBL1 is required for genome-wide PRC2 occupancy and function in cardiogenesis from human pluripotent stem cells
    (The Company of Biologists, 2021-07) Liu, Juli; Liu, Sheng; Han, Lei; Sheng, Yi; Zhang, Yucheng; Kim, Il-Man; Wan, Jun; Yang, Lei; Pediatrics, School of Medicine
    Polycomb repressive complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of the RNA-PRC2 interaction in human cardiogenesis remains elusive. Here, we found that human-specific heart brake lncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss of JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. In addition, ChIP-seq revealed reduced EED occupancy on 62 overlapped cardiogenic genes in HBL1−/− and JARID2−/− hPSCs, indicating that the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurs, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved ‘microRNA-1-JARID2’ axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis.
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    The Impaired Bioenergetics of Diabetic Cardiac Microvascular Endothelial Cells
    (Frontiers Media, 2021-05-14) Zhang, Haitao; Shen, Yan; Kim, Il-Man; Weintraub, Neal L.; Tang, Yaoliang; Anatomy, Cell Biology and Physiology, School of Medicine
    Diabetes causes hyperglycemia, which can create a stressful environment for cardiac microvascular endothelial cells (CMECs). To investigate the impact of diabetes on the cellular metabolism of CMECs, we assessed glycolysis by quantifying the extracellular acidification rate (ECAR), and mitochondrial oxidative phosphorylation (OXPHOS) by measuring cellular oxygen consumption rate (OCR), in isolated CMECs from wild-type (WT) hearts and diabetic hearts (db/db) using an extracellular flux analyzer. Diabetic CMECs exhibited a higher level of intracellular reactive oxygen species (ROS), and significantly reduced glycolytic reserve and non-glycolytic acidification, as compared to WT CMECs. In addition, OCR assay showed that diabetic CMECs had increased maximal respiration, and significantly reduced non-mitochondrial oxygen consumption and proton leak. Quantitative PCR (qPCR) showed no difference in copy number of mitochondrial DNA (mtDNA) between diabetic and WT CMECs. In addition, gene expression profiling analysis showed an overall decrease in the expression of essential genes related to β-oxidation (Sirt1, Acox1, Acox3, Hadha, and Hadhb), tricarboxylic acid cycle (TCA) (Idh-3a and Ogdh), and electron transport chain (ETC) (Sdhd and Uqcrq) in diabetic CMECs compared to WT CMECs. Western blot confirmed that the protein expression of Hadha, Acox1, and Uqcrq was decreased in diabetic CMECs. Although lectin staining demonstrated no significant difference in capillary density between the hearts of WT mice and db/db mice, diabetic CMECs showed a lower percentage of cell proliferation by Ki67 staining, and a higher percentage of cellular apoptosis by TUNEL staining, compared with WT CMECs. In conclusion, excessive ROS caused by hyperglycemia is associated with impaired glycolysis and mitochondrial function in diabetic CMECs, which in turn may reduce proliferation and promote CMEC apoptosis.