Publications

The human acetyltransferase complex HBO1 is implicated in cancer and developmental diseases. Here, we report the chromatin association mechanisms for scaffolding subunits of the complex, the JADE and BRPF paralogs. Structural and biochemical studies reveal a distinct paralog-specific engagement of the PZP domains of JADE1/2/3 and BRPF1 with the nucleosome. We show that only JADE1's PZP domain possesses a strong intrinsic DNA binding activity and binds to an almost entire histone H3 tail, forming a tight complex with the nucleosome. In contrast, the BRPF1 PZP domain's association with the nucleosome is a thousand-fold weaker due to its inability to engage with the large portion of H3 tail and a weak binding to DNA. These results suggest that the JADE1 PZP domain can protect the H3 tail from being modified, but the BRPF1 PZP domain cannot, which could contribute to functional differences observed in the HBO1 complexes. We demonstrate non-redundant functions of the paralogs throughout embryonic development and embryonic stem cell pluripotency, during cardiac cell differentiation, and in differentiated liver cells. Our findings provide insights into the diverse mechanisms of action of the HBO1 complex and help to understand how the scaffolding subunits mediate its different cellular functions.

Environmental disruption alters circadian clock gene expression, increasing the risk of adverse cardiac events and suggesting cardiomyocyte-specific circadian responses to external stimuli. Analyses of previously reported transcriptomic data revealed increased expression of Titin-cap (Tcap) in adult compared to embryonic myocytes and identified myosin light chain 2 (Myl2) as clock-controlled. Given cardiac sarcomeric roles of the encoded proteins, we hypothesized that extracellular cues driving postnatal cardiac maturation and hypertrophy influence time-of-day Tcap and Myl2 expression. Tcap induction was concomitant with neonatal myocyte binucleation, fetal gene suppression, and increased heart weight during the early phase of cardiac growth. Since norepinephrine stimulates β-adrenergic and α-adrenergic receptors, the latter driving clock-controlled transcriptional remodeling, phase‑response curves of the β‑adrenergic agonist isoproterenol (ISO) following α-adrenergic stimulation with phenylephrine (PE) were performed on neonatal rat ventricular myocytes (NRVM), revealing periodic myocyte hypertrophy and TCAP protein expression. PE entrenched ISO-mediated Tcap suppression, to which oscillatory Per2 and Myl2 transcription was impervious. Differential NRVM culture density revealed biomass-dependent changes in Per2 and Tcap transcription and hypertrophy timing. Hypoxia initiated myocyte atrophy and Bmal1-dependent Tcap transcription, reflected in decreased heart weight and increased Tcap expression in hypoxic neonatal rat hearts. Tcap depletion impaired Bmal1 and fetal hypertrophic gene expression, compromised hypoxia-mediated Myl2 transcriptional suppression, and aggravated hypoxia-induced atrophy. In summary, Tcap and Myl2 are circadian genes differentially influenced by environmental factors, including adrenergic stimulation, paracrine signaling, and O2 tension during postnatal cardiac maturation. These findings have implications for the distinct regulation of myocyte growth and maturation, by external cues during the postnatal period.

Heart failure (HF) is a major contributor to the global burden of cardiovascular disease. Current treatments for HF do not regenerate or restore cardiac muscle function, leaving cardiac transplantation as the only definitive treatment for end-stage HF. Subsequently, there is a tremendous need for alternative HF treatments as well as methods to effectively and selectively deliver those therapies to the heart. We have engineered an injectable reverse thermal gel (RTG) functionalized with carbon nanotubes (CNTs) to create a thermoresponsive conductive hydrogel or RTG-CNT. The RTG-CNT transitions from a liquid solution to a gel-based matrix upon reaching body temperature, a unique quality that allows for rapid injection of the liquid polymeric solution followed by gel localization in situ. Previously, we demonstrated the potential use of the RTG-CNT hydrogel for cardiac tissue engineering applications using three-dimensional (3D) cocultures of primary cardiac cells. Here, we performed a preclinical study to assess the biocompatibility of our RTG-CNT hydrogel in vivo by using hydrogel intracardial injection in a mouse model and in vitro by using 3D cultures of human-induced pluripotent stem cell-derived cardiomyocytes. In this report, we present compelling results that demonstrate the RTG-CNT hydrogel biocompatibility and its potential for use in cardiac tissue engineering applications.

Long QT syndrome (LQTS) is a channelopathy that predisposes affected individuals to ventricular arrhythmias and cardiac arrest. Here, a human induced pluripotent stem cell (hiPSC) line was generated from amniotic fluid cells (AFCs) of a 32-week fetus diagnosed with LQTS. Additionally, two iPSC lines were generated from peripheral blood mononuclear cells (PBMCs) of the fetus's healthy biological parents. Genome sequencing revealed that the fetus with LQTS carried a de novo KCNH2 variant, c.1898A > G (p.Asn633Ser). All three iPSC lines demonstrated normal morphology, karyotyping, and pluripotency. These iPSC lines provide a valuable in vitro model for LQTS caused by KCNH2 mutations.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-β, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. We sought to elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb after treatment with serum or the α-adrenergic agonist phenylephrine. Depletion of Bmal1 decreased the expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by micrococcal nuclease-quantitative PCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting natriuretic peptide B transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented phenylephrine-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

The human heart lacks significant regenerative capacity; thus, the solution to heart failure (HF) remains organ donation, requiring surgery and immunosuppression. The demand for constructed cardiac tissues (CCTs) to model and treat disease continues to grow. Recent advances in induced pluripotent stem cell (iPSC) manipulation, CRISPR gene editing, and 3D tissue culture have enabled a boom in iPSC-derived CCTs (iPSC-CCTs) with diverse cell types and architecture. Compared with 2D-cultured cells, iPSC-CCTs better recapitulate heart biology, demonstrating the potential to advance organ modeling, drug discovery, and regenerative medicine, though iPSC-CCTs could benefit from better methods to faithfully mimic heart physiology and electrophysiology. Here, we summarize advances in iPSC-CCTs and future developments in the vascularization, immunization, and maturation of iPSC-CCTs for study and therapy.

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H+) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo-electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H+ coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.

Myocardial infarction causes the loss of cardiomyocytes and the formation of cardiac fibrosis due to the activation of cardiac fibroblasts, leading to cardiac dysfunction and heart failure. Unfortunately, current therapeutic interventions can only slow the disease progression. Furthermore, they cannot fully restore cardiac function, likely because the adult human heart lacks sufficient capacity to regenerate cardiomyocytes. Therefore, intensive efforts have focused on developing therapeutics to regenerate the damaged heart. Several strategies have been intensively investigated, including stimulation of cardiomyocyte proliferation, transplantation of stem cell-derived cardiomyocytes, and conversion of fibroblasts into cardiac cells. Resident cardiac fibroblasts are critical in the maintenance of the structure and contractility of the heart. Fibroblast plasticity makes this type of cells be reprogrammed into many cell types, including but not limited to induced pluripotent stem cells, induced cardiac progenitor cells, and induced cardiomyocytes. Fibroblasts have become a therapeutic target due to their critical roles in cardiac pathogenesis. This review summarizes the reprogramming of fibroblasts into induced pluripotent stem cell-derived cardiomyocytes, induced cardiac progenitor cells, and induced cardiomyocytes to repair a damaged heart, outlines recent findings in utilizing fibroblast-derived cells for heart regeneration, and discusses the limitations and challenges.

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.