Epigenetic inheritance of gene expression states enables a single genome to maintain distinct cellular identities. How histone modifications contribute to this process remains unclear. Using global chromatin perturbations and local, time-controlled modulation of transcription, we establish the existence of epigenetic memory of transcriptional activation for genes that can be silenced by the Polycomb group. This property emerges during cell differentiation and allows genes to be stably switched following a transient transcriptional stimulus. This transcriptional memory state at Polycomb targets operates in cis; however, rather than relying solely on read-and-write propagation of histone modifications, the memory is also linked to the strength of activating inputs opposing Polycomb proteins and therefore varies with the cellular context. Our data and computational simulations suggest a model whereby transcriptional memory arises from double-negative feedback between Polycomb-mediated silencing and active transcription. Transcriptional memory at Polycomb targets thus depends not only on histone modifications but also on the gene-regulatory network and underlying identity of a cell.
Rationale: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1 + progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. Objective: To determine the role of pulmonary PW1 + progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. Methods and Results: We investigated their contribution during chronic hypoxia–induced vascular remodeling in Pw1 nLacZ+/− mouse expressing β-galactosidase in PW1 + cells and in differentiated cells derived from PW1 + cells. PW1 + progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1 + cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1 + cells and the proportion of β-gal + vascular SMC were increased, indicating a recruitment of PW1 + cells and their differentiation into vascular SMC during early chronic hypoxia–induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1 + cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal + SMC were not derived from circulating bone marrow–derived PW1 + progenitor cells, confirming a resident origin of the recruited PW1 + cells. The number of pulmonary PW1 + cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. Conclusions: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension–associated vascular remodeling.
BackgroundArrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D) is an inherited cardiomyopathy mainly caused by heterozygous desmosomal gene mutations, the major gene being PKP2. The genetic cause remains unknown in ~50% of probands with routine desmosomal gene screening. The aim of this study was to assess the diagnostic accuracy of whole exome sequencing (WES) in ARVC/D with negative genetic testing.MethodsWES was performed in 22 patients, all without a mutation identified in desmosomal genes. Putative pathogenic variants were screened in 96 candidate genes associated with other cardiomyopathies/channelopathies. The sequencing coverage depth of PKP2, DSP, DSG2, DSC2, JUP and TMEM43 exons was compared to the mean coverage distribution to detect large insertions/deletions. All suspected deletions were verified by real-time qPCR, Multiplex-Ligation-dependent-Probe-Amplification (MLPA) and cGH-Array. MLPA was performed in 50 additional gene-negative probands.ResultsCoverage-depth analysis from the 22 WES data identified two large heterozygous PKP2 deletions: one from exon 1 to 14 and one restricted to exon 4, confirmed by qPCR and MLPA. MLPA identified 2 additional PKP2 deletions (exon 1–7 and exon 1–14) in 50 additional probands confirming a significant frequency of large PKP2 deletions (5.7%) in gene-negative ARVC/D. Putative pathogenic heterozygous variants in EYA4, RBM20, PSEN1, and COX15 were identified in 4 unrelated probands.ConclusionA rather high frequency (5.7%) of large PKP2 deletions, undetectable by Sanger sequencing, was detected as the cause of ARVC/D. Coverage-depth analysis through next-generation sequencing appears accurate to detect large deletions at the same time than conventional putative mutations in desmosomal and cardiomyopathy-associated genes.
Aims Desmoglein-2 (DSG2) mutations, which encode a heart-specific cadherin crucial for desmosomal adhesion, are frequent in arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). DSG2 mutations have been associated with higher risk of biventricular involvement. Among DSG2 mutations, mutations of the inhibitory propeptide consensus cleavage-site (Arg-X-Arg/Lys-Arg), are particularly frequent. In the present work, we explored the functional consequences of DSG2 propeptide cleavage site mutations p.Arg49His, p.Arg46Trp, and p.Arg46Gln on localization, adhesive properties, and desmosome incorporation of DSG2. Methods and results We studied the expression of mutant-DSG2 in human heart and in epithelial and cardiac cellular models expressing wild-type or mutant (p.Arg49His, p.Arg46Trp, and p.Arg46Gln) proDSG2-GFP fusion proteins. The consequences of the p.Arg46Trp mutation on DSG2 adhesiveness were studied by surface plasmon resonance. Incorporation of mutant p.Arg46Trp DSG2 into desmosomes was studied under low-calcium culture conditions and cyclic mechanical stress. We demonstrated in human heart and cellular models that all three mutations prevented N-terminal propeptide cleavage, but did not modify intercellular junction targeting. Surface plasmon resonance experiments showed a propeptide-dependent loss of interaction between the cadherin N-terminal extracellular 1 (EC1) domains. Additionally, proDSG2 mutant proteins were abnormally incorporated into desmosomes under low-calcium culture conditions or following mechanical stress. This was accompanied by an epidermal growth factor receptor-dependent internalization of proDSG2, suggesting increased turnover of unprocessed proDSG2. Conclusion Our results strongly suggest weakened desmosomal adhesiveness due to abnormal incorporation of uncleaved mutant proDSG2 in cellular stress conditions. These results provide new insights into desmosomal cadherin regulation and ARVC/D pathophysiology, in particular, the potential role of mechanical stress on desmosomal dysfunction.
Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with -galactosidase adenovirus (Ϫ71%; P Ͻ 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions. lysophosphatidate; lipid phosphate phosphatase; phenotypic modulation; neointimal formation; MAPK VASCULAR SMOOTH MUSCLE CELLS (VSMCs) are known to be one of the cell types that exist along a continuum of phenotypes. A differentiated phenotype with a contractile function is predominant in mature arteries, whereas a phenotypic modulation (22), characterized by a switch towards a synthetic profile, occurs in vascular injury conditions such as atherosclerosis or restenosis following percutaneous transluminal angioplasty.Lysophosphatidic acid (LPA), a potent bioactive glycerophospholipid, plays an important role in VSMCs dedifferentiation characterized in vitro by proliferation and migration (13, 31) and in vivo by neointima formation (39). LPA concentration in the serum results from a balance between its synthesis by autotaxin and/or A-type phospholipase (2), at least in part after platelet activation, and degradation by the lipid phosphate phosphatases (LPPs; Ref. 6). In a global way, LPA is considered as a pathophysiological mediator of vascular cell function involved in many cardiovascular diseases such as atherothrombosis (25, 28) and hypertension (36). Several studies contributed to shed light on the mechanisms underlying the effects of LPA. In the VSMCs, it binds to specific G-protein-coupled receptors, LPA1 to LPA4 (13, 18), activating many downstream pathways involving signaling proteins such as RhoA and Erk1/2 (1, 18, 23) and transcription factors such as response element binding protein and serum response factor leading to Egr-1 expression (8), which promotes e...
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