Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Indexación: Web of Science; Scopus.Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotencyhttp://mcb.asm.org/content/36/4/61

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events

Molecular characterization of human osteoblast-derived extracellular vesicle mRNA using next-generation sequencing

Extracellular vesicles (EVs) are membrane-bound intercellular communication vehicles that transport proteins, lipids and nucleic acids with regulatory capacity between cells. RNA profiling using microarrays and sequencing technologies has revolutionized the discovery of EV-RNA content, which is crucial to understand the molecular mechanism of EV function. Recent studies have indicated that EVs are enriched with specific RNAs compared to the originating cells suggestive of an active sorting mechanism. Here, we present the comparative transcriptome analysis of human osteoblasts and their corresponding EVs using next-generation sequencing. We demonstrate that osteoblast-EVs are specifically depleted of cellular mRNAs that encode proteins involved in basic cellular activities, such as cytoskeletal functions, cell survival and apoptosis. In contrast, EVs are significantly enriched with 254 mRNAs that are associated with protein translation and RNA processing. Moreover, mRNAs enriched in EVs encode proteins important for communication with the neighboring cells, in particular with osteoclasts, adipocytes and hematopoietic stem cells. These findings provide the foundation for understanding the molecular mechanism and function of EV-mediated interactions between osteoblasts and the surrounding bone microenvironment

Inhibition of PARP Sensitizes Chondrosarcoma Cell Lines to Chemo- and Radiotherapy Irrespective of the IDH1 or IDH2 Mutation Status

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutationsin isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate(D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP)inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repairand PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma celllines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with thePARP inhibitor talazoparib were examined for dose–response relationships, as well as underlyingcell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- orradiotherapy to evaluate potential synergy. Cell lines treated long termwith an inhibitor normalizingD-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparibsensitivity was variable and irrespective of IDH mutation status. All cell lines expressed AtaxiaTelangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation)capacity, homologous recombination, andO-6-methylguanine-DNAmethyltransferase (MGMT) expression.Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition.This study suggests that talazoparib combined with temozolomide or radiation are promisingtherapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset ofchondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARPinhibitor sensitivity is multifactorial in chondrosarcoma.Toxicolog

Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34 + HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34 + cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders

Identification of osteolineage cell-derived extracellular vesicle cargo implicated in hematopoietic support

Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs-altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study giv

Hereditary cancer registries improve the care of patients with a genetic predisposition to cancer: contributions from the Dutch Lynch syndrome registry

The Dutch Hereditary Cancer Registry was established in 1985 with the support of the Ministry of Health (VWS). The aims of the registry are: (1) to promote the identification of families with hereditary cancer, (2) to encourage the participation in surveillance programs of individuals at high risk, (3) to ensure the continuity of lifelong surveillance examinations, and (4) to promote research, in particular the improvement of surveillance protocols. During its early days the registry provided assistance with family investigations and the collection of medical data, and recommended surveillance when a family fulfilled specific diagnostic criteria. Since 2000 the registry has focused on family follow-up, and ensuring the quality of surveillance programs and appropriate clinical management. Since its founding, the registry has identified over 10,000 high-risk individuals with a diverse array of hereditary cancer syndromes. All were encouraged to participate in prevention programmes. The registry has published a number of studies that evaluated the outcome of surveillance protocols for colorectal cancer (CRC) in Lynch syndrome, as well as in familial colorectal cancer. In 2006, evaluation of the effect of registration and colonoscopic surveillance on the mortality rate associated with colorectal cancer (CRC) showed that the policy led to a substantial decrease in the mortality rate associated with CRC. Following discovery of MMR gene defects, the first predictive model that could select families for genetic testing was published by the Leiden group. In addition, over the years the registry has produced many cancer risk studies that have helped to develop appropriate surveillance protocols. Hereditary cancer registries in general, and the Lynch syndrome registry in particular, play an important role in improving the clinical management of affected families.Hereditary cancer genetic

Bariatric surgery for hypothalamic obesity in craniopharyngioma patients: a retrospective, matched case-control study

Context: Craniopharyngioma is a sellar tumor associated with high rates of pituitary deficiencies (similar to 98%) and hypothalamic obesity (similar to 50%).Objective: This work aims to determine the efficacy regarding long-term weight loss after bariatric surgery in obese craniopharyngioma patients with hypothalamic dysfunction.Methods: This retrospective, case-control, multicenter, international study included obese craniopharyngioma patients (N=16; of whom 12 are women) with a history of bariatric surgery (12 Roux-en-Y gastric bypass, 4 sleeve gastrectomy; median age 21 years [range, 15-52 years], median follow-up 5.2 years [range, 2.0-11.3 years]) and age/sex/surgery/body mass index-matched obese controls (N=155). Weight loss and obesity-related comorbidities up to 5 years after bariatric surgery were compared and changes in hormonal replacement therapy evaluated.Results: Mean weight loss at 5-year follow-up was 22.0% (95% CI, 16.1%-27.8%) in patients vs 29.5% (95% CI, 28.0%-30.9%) in controls (P=.02), which was less after Roux-en-Y gastric bypass (22.7% [16.9%-28.5%] vs 32.0% [30.4%-33.6%]; P=.003) but at a similar level after sleeve gastrectomy (21.7% [-1.8% to 45.2%] vs 21.8% [18.2%-25.5%]; P=.96). No major changes in endocrine replacement therapy were observed after surgery. One patient died (unknown cause). One patient had long-term absorptive problems.Conclusion: Obese patients with craniopharyngioma had a substantial mean weight loss of 22% at 5-year follow-up after bariatric surgery, independent of type of bariatric surgery procedure. Weight loss was lower than in obese controls after Roux-en-Y gastric bypass. Bariatric surgery appears to be effective and relatively safe in the treatment of obese craniopharyngioma patients.Development and application of statistical models for medical scientific researc

Standardized postnatal management of infants with congenital diaphragmatic hernia in Europe: The CDH EURO Consortium Consensus - 2015 Update

In 2010, the congenital diaphragmatic hernia (CDH) EURO Consortium published a standardized neonatal treatment protocol. Five years later, the number of participating centers has been raised from 13 to 22. In this article the relevant literature is updated, and consensus has been reached between the members of the CDH EURO Consortium. Key updated recommendations are: (1) planned delivery after a gestational age of 39 weeks in a high-volume tertiary center; (2) neuromuscular blocking agents to be avoided during initial treatment in the delivery room; (3) adapt treatment to reach a preductal saturation of between 80 and 95% and postductal saturation >70%; (4) target PaCO2 to be between 50 and 70 mm Hg; (5) conventional mechanical ventilation to be the optimal initial ventilation strategy, and (6) intravenous sildenafil to be considered in CDH patients with severe pulmonary hypertension. This article represents the current opinion of all consortium members in Europe for the optimal neonatal treatment of CDH