The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in ...end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist.
We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability.
Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.
DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted ...transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding.
Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation.
Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.
Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another ...well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogeneous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.
This report describes the use of late‐outgrowth endothelial progenitor cells (L‐EPCs), as a cellular substrate for the generation of induced pluripotent stem cells (iPSCs). A protocol was developed ...that allows the reliable isolation of L‐EPCs from peripheral blood mononuclear cell preparations, including frozen samples. L‐EPCs grew clonally, were proliferative, were bankable, and had karyotypes representative of their donor. L‐EPCs reprogrammed to iPSCs with good efficiencies and the iPSCs had karyotypes representative of the L‐EPCs used to generate them. This work identifies L‐EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.
Induced pluripotent stem cells (iPSCs) have the potential to generate patient‐specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late‐outgrowth endothelial progenitor cells (L‐EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L‐EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof‐of‐principle for clinical applications we generated EPC‐iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L‐EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L‐EPC reprogramming allowed for the reliable generation of iPSCs in a 96‐well format, which is compatible with high‐throughput platforms. Array comparative genome hybridization analysis of L‐EPCs versus donor‐matched circulating monocytes demonstrated that L‐EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC‐iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L‐EPC line. This work identifies L‐EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.
Rapidly advancing high-throughput sequencing technology is now bringing attention to many basic biological aspects of the human genome. DNA methylation refers to the epigenetic modification of ...cytosine nucleotides by a methyl group that occurs throughout the genome. Owing to its significant influence on protein-–DNA interactions and subsequent gene-expression control, some scientists call methylated-cytosines '‘the 5th nucleotide ’. We recently reported the first evidence of differential DNA methylation in human heart failure. Altered DNA methylation and a change in the expression of proximal genes have also been demonstrated in atherosclerotic plaques. For other diseases such as psychosis and cancer, the role of DNA methylation on disease pathogenesis and progression has already been shown and forms the target for new drug therapy. Understanding this aspect of disease biology may therefore contribute to the heart failure drug discovery pipeline. In this article, we summarize the basic biology of DNA methylation and discuss its implications in complex diseases such as heart failure.
The human variome: genomic and epigenomic diversity Choy, Munkit; Movassagh, Mehregan; Foo, Roger
EMBO molecular medicine,
October 2011, 2011-Oct, 2011-10-00, 20111001, Letnik:
3, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Mechanistically, disease‐associated SNPs may be found in DNA regulatory regions where the genetic variation affects cognate binding of transcription factor complexes, effecting on the expression of ...disease‐relevant genes (Harismendy et al, 2011). ...variation in the methylation of a DNA regulatory region (either increased or decreased methylation) can also alter the binding of transcription factor complexes and regulate both distal and proximal gene expression (Choy et al, 2010; Jones & Takai, 2001; Phillips & Corces, 2009; Weaver et al, 2004). On the basis that increased DNA methylation may be a means of regulating transcription factor complex binding and therefore mark genomic regions that are important to control by methylation (Choy et al, 2010; Jones & Takai, 2001), our findings support the notion that sites of heterogeneous genetic variation in one subpopulation are functionally relevant to the corresponding subpopulation, but sites of heterogeneous genetic variation of a different subpopulation, are not.
The proneural protein neurogenin (XNGNR1) drives differentiation of primary neurons in combination with the cyclin‐dependent kinase (Cdk) inhibitor Xic1. Differentiation is inhibited by Notch ...signalling, resulting in a scattered neuronal distribution. Here we show that Notch signalling regulates the level of Xic1 transcription, yet this does not correlate with Notch's ability to perturb the cell cycle. Instead, Notch may regulate Xic1 levels to control its differentiation function directly, which is required in parallel with XNGNR1 to promote primary neurogenesis. Indeed, Notch‐mediated repression of both XNGNR1 and Xic1 must be relieved for neuronal differentiation to occur. Interestingly, although Xic1 is required for XNGNR1‐mediated neurogenesis, it is not required for XNGNR1‐mediated upregulation of Delta, allowing establishment of the negative feedback loop involved in lateral inhibition. Therefore, Notch targets Cdk inhibitor expression to regulate differentiation of primary neurons, and its effects on the cell cycle may be of secondary importance.
BACKGROUND: The epigenomes of healthy and diseased human hearts were recently examined by genome-wide DNA methylation profiling. Repetitive elements, heavily methylated in post-natal tissue, have ...variable methylation profiles in cancer but methylation of repetitive elements in the heart has never been examined. RESULTS: We analyzed repetitive elements from all repeat families in human myocardial samples, and found that satellite repeat elements were significantly hypomethylated in end-stage cardiomyopathic hearts relative to healthy normal controls. Satellite repeat elements are almost always centromeric or juxtacentromeric, and their overexpression correlates with disease aggressiveness in cancer. Similarly, we found that hypomethylation of satellite repeat elements correlated with up to 27-fold upregulation of the corresponding transcripts in end-stage cardiomyopathic hearts. No other repeat family exhibited differential methylation between healthy and cardiomyopathic hearts, with the exception of the Alu element SINE1/7SL, for which a modestly consistent trend of increased methylation was observed. CONCLUSIONS: Satellite repeat element transcripts, a form of non-coding RNA, have putative functions in maintaining genomic stability and chromosomal integrity. Further studies will be needed to establish the functional significance of these non-coding RNAs in the context of heart failure.
Cardiac hypertrophy is a growth response of the heart to increased hemodynamic demand or damage. Accompanying this heart enlargement is a remodeling of Ca 2+ signaling. Due to its fundamental role in ...controlling cardiomyocyte contraction during every heartbeat, modifications in Ca 2+ fluxes significantly impact on cardiac output and facilitate the development of arrhythmias. Using cardiomyocytes from spontaneously hypertensive rats (SHRs), we demonstrate that an increase in Ca 2+ release through inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) contributes to the larger excitation contraction coupling (ECC)-mediated Ca 2+ transients characteristic of hypertrophic myocytes and underlies the more potent enhancement of ECC-mediated Ca 2+ transients and contraction elicited by InsP 3 or endothelin-1 (ET-1). Responsible for this is an increase in InsP 3 R expression in the junctional sarcoplasmic reticulum. Due to their close proximity to ryanodine receptors (RyRs) in this region, enhanced Ca 2+ release through InsP 3 Rs served to sensitize RyRs, thereby increasing diastolic Ca 2+ levels, the incidence of extra-systolic Ca 2+ transients, and the induction of ECC-mediated Ca 2+ elevations. Unlike the increase in InsP 3 R expression and Ca 2+ transient amplitude in the cytosol, InsP 3 R expression and ECC-mediated Ca 2+ transients in the nucleus were not altered during hypertrophy. Elevated InsP 3 R2 expression was also detected in hearts from human patients with heart failure after ischemic dilated cardiomyopathy, as well as in aortic-banded hypertrophic mouse hearts. Our data establish that increased InsP 3 R expression is a general mechanism that underlies remodeling of Ca 2+ signaling during heart disease, and in particular, in triggering ventricular arrhythmia during hypertrophy.
Aims Cyclin-dependent kinase inhibitors (CDKIs) play a critical role in negatively regulating the proliferation of cardiomyocytes, although their role in cardiac differentiation remains largely ...undetermined. We have shown that the most prominent CDKI in Xenopus, p27Xic1(Xic1), plays a role in neuronal and myotome differentiation beyond its ability to arrest the cell cycle. Thus, we investigated whether it plays a similar role in cardiomyocyte differentiation. Methods and results Xenopus laevis embryos were sectioned, and whole-mount antibody staining and immunofluorescence studies were carried out to determine the total number and percentage of differentiated cardiomyocytes in mitosis. Capped RNA and/or translation-blocking Xic1 morpholino antisense oligonucleotides (Xic1Mo) were microinjected into embryos, and their role on cardiac differentiation was assessed by in situ hybridization and/or PCR. We show that cell-cycling post-gastrulation is not essential for cardiac differentiation in Xenopus embryos, and conversely that some cells can express markers of cardiac differentiation even when still in cycle. A targeted knock-down of Xic1 protein by Xic1Mo microinjection decreases the expression of markers of cardiac differentiation, which can be partially rescued by co-injection of full-length Xic1 RNA, demonstrating that Xic1 is essential for heart formation. Furthermore, using deleted and mutant forms of Xic1, we show that neither its abilities to inhibit the cell cycle nor the great majority of CDK kinase activity are essential for Xic1’s function in cardiomyocyte differentiation, an activity that resides in the N-terminus of the molecule. Conclusion Altogether, our results demonstrate that the CDKI Xic1 is required in Xenopus cardiac differentiation, and that this function is localized at its N-terminus, but it is distinct from its ability to arrest the cell cycle and inhibit overall CDK kinase activity. Hence, these results suggest that CDKIs play an important direct role in driving cardiomyocyte differentiation in addition to cell-cycle regulation.
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