Paul Delgado-Olguin

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo.

MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR‐146a and miR‐146b are induced in endothelial cells upon exposure to pro‐inflammatory cytokines. Despite the rapid transcriptional induction of the miR‐146a/b loci, which is in part mediated by EGR‐3, miR‐146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down‐regulation of inflammatory gene expression. We demonstrate that miR‐146 negatively regulates inflammation. Over‐expression of miR‐146a blunts endothelial activation, while knock‐down of miR‐146a/b in vitro or deletion of miR‐146a in mice has the opposite effect. MiR‐146 represses the pro‐inflammatory NF‐κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR‐146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro‐inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases.

Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis

Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown. The Polycomb histone methyltransferase Ezh2 stabilizes transcription by depositing repressive marks during development that persist into adulthood, but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor gene Six1, which functions in cardiac progenitor cells but is stably silenced upon cardiac differentiation. Deletion of Ezh2 in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable gene expression and homeostasis in the postnatal heart. Our results suggest that epigenetic dysregulation in embryonic progenitor cells is a predisposing factor for adult disease and dysregulated stress responses.

Chromatin remodelling complex dosage modulates transcription factor function in heart development

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His–Purkinje network

Rapid electrical conduction in the His–Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His–Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction–forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Ezh2 regulates anteroposterior axis specification and proximodistal axis elongation in the developing limb

Specification and determination (commitment) of positional identities precedes overt pattern formation during development. In the limb bud, it is clear that the anteroposterior axis is specified at a very early stage and is prepatterned by the mutually antagonistic interaction between Gli3 and Hand2. There is also evidence that the proximodistal axis is specified early and determined progressively. Little is known about upstream regulators of these processes or how epigenetic modifiers influence axis formation. Using conditional mutagenesis at different time points, we show that the histone methyltransferase Ezh2 is an upstream regulator of anteroposterior prepattern at an early stage. Mutants exhibit posteriorised limb bud identity. During later limb bud stages, Ezh2 is essential for cell survival and proximodistal segment elongation. Ezh2 maintains the late phase of Hox gene expression and cell transposition experiments suggest that it regulates the plasticity with which cells respond to instructive positional cues.

CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

CTCF nuclear factor regulates many aspects of gene expression, largely as a transcriptional repressor or via insulator function. Its roles in cellular differentiation are not clear. Here we show an unexpected role for CTCF in myogenesis. Ctcf is expressed in myogenic structures during mouse and zebrafish development. Gain- and loss-of-function approaches in C2C12 cells revealed CTCF as a modulator of myogenesis by regulating muscle-specific gene expression. We addressed the functional connection between CTCF and myogenic regulatory factors (MRFs). CTCF enhances the myogenic potential of MyoD and myogenin and establishes direct interactions with MyoD, indicating that CTCF regulates MRF-mediated muscle differentiation. Indeed, CTCF modulates functional interactions between MyoD and myogenin in co-activation of muscle-specific gene expression and facilitates MyoD recruitment to a muscle-specific promoter. Finally, ctcf loss-of-function experiments in zebrafish embryos revealed a critical role of CTCF in myogenic development and linked CTCF to broader aspects of development via regulation of Wnt signaling. We conclude that CTCF modulates MRF functional interactions in the orchestration of myogenesis.

Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function.

Human dilated cardiomyopathy (DCM) manifests as a profound reduction in biventricular cardiac function that typically progresses to death or cardiac transplantation. There is no effective mechanism-based therapy currently available for DCM, in part because the transduction of mechanical load into dynamic changes in cardiac contractility (termed mechanotransduction) remains an incompletely understood process during both normal cardiac function and in disease states. Here we show that the mechanoreceptor protein integrin-linked kinase (ILK) mediates cardiomyocyte force transduction through regulation of the key calcium regulatory protein sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase isoform 2a (SERCA-2a) and phosphorylation of phospholamban (PLN) in the human heart. A non-oncogenic ILK mutation with a synthetic point mutation in the pleckstrin homology-like domain (ILK(R211A)) is shown to enhance global cardiac function through SERCA-2a/PLN. Thus, ILK serves to link mechanoreception to the dynamic modulation of cardiac contractility through a previously undiscovered interaction with the functional SERCA-2a/PLN module that can be exploited to rescue impaired mechanotransduction in DCM.

Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculature

Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.

Chromatin structure of pluripotent stem cells and induced pluripotent stem cells.

Pluripotent embryonic stem (ES) cells are specialized cells with a dynamic chromatin structure, which is intimately connected with their pluripotency and physiology. In recent years somatic cells have been reprogrammed to a pluripotent state through over-expression of a defined set of transcription factors. These cells, known as induced pluripotent stem (iPS) cells, recapitulate ES cell properties and can be differentiated to apparently all cell lineages, making iPS cells a suitable replacement for ES cells in future regenerative medicine. Chromatin modifiers play a key function in establishing and maintaining pluripotency, therefore, elucidating the mechanisms controlling chromatin structure in both ES and iPS cells is of utmost importance to understanding their properties and harnessing their therapeutic potential. In this review, we discuss recent studies that provide a genome-wide view of the chromatin structure signature in ES cells and iPS cells and that highlight the central role of histone modifiers and chromatin remodelers in pluripotency maintenance and induction.