Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPS state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPS cells display similar characteristics to Oct4/Sox2/Klf4/Myc-iPS cells, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can co-operate in a powerful way to reprogram somatic cells to pluripotency.
In the adult heart, a variety of stresses induce re-expression of a fetal gene program in association with myocyte hypertrophy and heart failure. Here we show that histone deacetylase-2 (Hdac2) regulates expression of many fetal cardiac isoforms. Hdac2 deficiency or chemical histone deacetylase (HDAC) inhibition prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp5f) resulting in constitutive activation of glycogen synthase kinase 3beta (Gsk3beta) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, Hdac2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3beta. Chemical inhibition of activated Gsk3beta allowed Hdac2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that Hdac2 is an important molecular target of HDAC inhibitors in the heart and that Hdac2 and Gsk3beta are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.
The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar Type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging Type I cells. In vivo, Type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here, we show that Hopx becomes restricted to Type I cells during development. However, unexpectedly, lineage-labeled Hopx+ cells both proliferate and generate Type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx+ Type I cells generate organoids composed of Type I and Type II cells, a process modulated by TGFβ signaling. These findings demonstrate unanticipated plasticity of Type I cells and a bi-directional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single cell culture.
Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b -/-embryoid bodies. While GSK-3α-deficient mice were born without a cardiac phenotype, no live-born Gsk3b -/-pups were recovered. The Gsk3b -/-embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation -GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3α and GSK-3β in the heart using loss-of-function approaches, implicate GSK-3β as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.
Summary Regulation of chromatin structure via histone modification has received intense recent attention. Here, we demonstrate that the chromatin modifying enzyme histone deacetylase 2 (Hdac2) functions with a small homeodomain factor, Hopx, to mediate deacetylation of Gata4, which is expressed by cardiac progenitor cells and plays critical roles in the regulation of cardiogenesis. In the absence of Hopx and Hdac2 in mouse embryos, Gata4 hyperacetylation is associated with a marked increase in cardiac myocyte proliferation, up-regulation of Gata4 target genes, and peri-natal lethality. Hdac2 physically interacts with Gata4, and this interaction is stabilized by Hopx. The ability of Gata4 to transactivate cell cycle genes is impaired by Hopx/Hdac2-mediated deacetylation and this effect is abrogated by loss of Hdac2-Gata4 interaction. These results suggest that Gata4 is a non-histone target of Hdac2-mediated deacetylation and that Hdac2, Hopx and Gata4 coordinately regulate cardiac myocyte proliferation during embryonic development.
Class I and II histone deacetylases (HDACs) play vital roles in regulating cardiac development, morphogenesis, and hypertrophic responses. Although the roles of Hdac1 and Hdac2, class I HDACs, in cardiac hyperplasia, growth, and hypertrophic responsiveness have been reported, the role in the heart of Hdac3, another class I HDAC, has been less well explored. Here we report that myocyte-specific overexpression of Hdac3 in mice results in cardiac abnormalities at birth. Hdac3 overexpression produces thickening of ventricular myocardium, especially the interventricular septum, and reduction of both ventricular cavities in newborn hearts. Our data suggest that increased thickness of myocardium in Hdac3-transgenic (Hdac3-Tg) mice is due to increased cardiomyocyte hyperplasia without hypertrophy. Hdac3 overexpression inhibits several cyclin-dependent kinase inhibitors, including Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, and Cdkn2c. Hdac3-Tg mice did not develop cardiac hypertrophy at 3 months of age, unlike previously reported Hdac2-Tg mice. Further, Hdac3 overexpression did not augment isoproterenol-induced cardiac hypertrophy when compared with wild-type littermates. These findings identify Hdac3 as a novel regulator of cardiac myocyte proliferation during cardiac development.Histone acetyl transferases and histone deacetylases (HDACs) 3 are critical regulators of chromatin remodeling (1). Modifications of core histones, around which DNA is packaged, play prominent roles in the regulation of gene expression (2). For instance, core histones are acetylated by histone acetyl transferases, leading to unwinding and relaxation of the chromatin structure and subsequent gene activation. This enzymatic acetylation step is reversed by HDAC-dependent deacetylation, leading to chromatin condensation and gene repression (3). Histone modifications provide marks for interactions with transcriptional coactivator and repressor proteins to regulate gene expression (4). The mammalian HDACs are classified into four subfamilies based on their highly conserved homologous sequences and functional similarities (3). Class I HDACs include HDAC1, -2, -3, and -8; class II HDACs include HDAC4, -5, -6, -7, -9, and -10; class III HDACs are known as sirtuins (5); and the lone class IV HDAC is HDAC11 (6). Conventional HDAC inhibitors, such as trichostatin A, inhibit both class I and class II HDACs (7).Various class I and II Hdac gene deletion and overexpression analyses in mice have suggested important roles for histone modification and HDAC function in cardiac development and in the regulation of cardiac hypertrophy (8 -13). For example, class II HDACs, including Hdac5 and Hdac9, act as negative regulators of cardiac growth (14). Inactivation of either Hdac5 or Hdac9 results in mice that are sensitive to hypertrophic stress, whereas the simultaneous loss of both Hdac5 and Hdac9 produces mice with grossly enlarged hearts (15). Loss of Hdac7 results in abnormal thinning of myocardial walls of the ventricular chambers and dilation of atria (16). Deletion of t...
Background: Histone-modifying genes play critical roles in the pathogenesis of human congenital heart disease. Results: HDAC3 recruits PRC2 complex to mediate epigenetic silencing of TGF-1 in a deacetylase-independent manner within second heart field progenitor cells. Conclusion: HDAC3-mediated epigenetic silencing of TGF-1 is required for normal heart development. Significance: This is the first report of HDAC-mediated epigenetic regulation of second heart field development.
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