DNA Demethylation in Pluripotency and Reprogramming: The Role of Tet Proteins and Cell Division

Cytosine methylation is found in the genomes of many plants and animals and has been associated with transcriptional silencing in mammals. At critical stages in embryo development, when cellular potential is reset, DNA methylation is lost in a series of “sequential waves.” The mechanism underlying this is controversial and complex. Several new reports now suggest that TET enzymes and cell division are important for these in vivo transitions as well as for experimentally induced reprogramming

Harnessing bioluminescence for drug discovery and epigenetic research

The naturally occurring phenomenon of bioluminescence has intrigued on-lookers for decades and is now being developed as a powerful tool for medical research and preclinical imaging. Luciferase enzymes emit light upon substrate encounter, enabling their activity to be visualised and dynamically tracked. By inserting luciferase genes into specific sites in the genome, it is possible to engineer reporters to monitor gene expression in its native context, and to detect epigenetic change in vivo. Endogenous bioluminescent reporters provide a highly sensitive, quantitative read-out of gene expression that is both well suited to longitudinal studies and can be adapted for high-throughput drug screens. In this article we outline some of the applications and benefits of bioluminescent reporters for epigenetic research, with a particular focus on revealing new therapeutic options for treating genetic and epigenetic disorders

Expression of Functional MHC Class II Molecules By a Mouse Pro-B Cell Clone

We describe here the G12 pro-B cell clone that has been isolated from an IL-7 transgenic mouse. This clone has the phenotype B220+, BP-1+ , HSA +, CD43+ λ5+ , and CD25-, and has its Ig locus in a germline configuration. G12 cells spontaneously express cell-surface MHC class II molecules, although to a much lesser extent than the mature M12.4.1 B-cell lymphoma. G12 cells can process and present the native Hen Egg Lysozyme (HEL) to an MHC class II-restricted T-cell hybridoma. The efficiency of presentation is inferior to that obtained with M12.4.1 cells. This is the first report where a pro-B cell can serve as APC in an MHC class II-restricted presentation

Characterization of Murine Thymic Stromal-Cell Lines Immortalized by Temperature-Sensitive Simian Virus 40 Large T or Adenovirus 5 E1a

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined

Centromeric Repositioning of Coreceptor Loci Predicts Their Stable Silencing and the CD4/CD8 Lineage Choice

The differentiation of CD4+ CD8+ double positive (DP) thymocytes requires the irreversible choice between two alternative lineages, distinguished by the mutually exclusive expression of either CD4 or CD8. Differentiating DP cells transiently down-regulate both CD4 and CD8, and this has complicated the debate whether the mechanism of CD4/CD8 lineage choice is instructive, stochastic/selective, or more complex in nature. Using fluorescence in situ hybridization, we show that the stable silencing of coreceptor loci, and ultimately lineage choice, is predicted by the spatial repositioning of coreceptor alleles to centromeric heterochromatin domains. These data provide evidence that lineage-specific developmental programs are established early during the transition from the DP to the single positive stage

Acute Exercise Activates Pulmonary eNOS and Lowers Pulmonary Pressure in Rats with Pulmonary Arterial Hypertension

poster abstractNO-dependent arterial relaxation is impaired in pulmonary arterial hypertension (PAH). Exercise may be beneficial in PAH, just as it is for systemic vascular disease, via upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. However, exercise-induced cardiac stress in PAH could also promote detrimental RV inflammation. We investigated pulmonary pressure and eNOS, as well inflammatory indicators in the RV, following a single 45 min run bout at moderate intensity in a rat model of PAH. Male Sprague-Dawley rats received either monocrotaline to induce PAH, or saline, for healthy controls. A subset of PAH and healthy controls performed 4 wks of progressive TM familiarization (15-30min, 8-20 m/min) in preparation for their final 45 min run @ 75% of VO2max. Immediately following the run, RV systolic pressure was measured and RV and lung tissues were harvested and cryofixed. eNOS and phosphorylated (at Ser1177) eNOS (p-eNOS) was measured via immunoblotting in lung homogenates and expressed normalized to vinculin. Immunofluorescence for inflammatory markers CD45/68 in cryofixed RV sections evaluated the acute inflammatory response to exercise. MCT reduced VO2max and caused RV hypertrophy (expressed as RV/LV+septum) as consistent with this model. RVSP (normalized by systemic BP) was lower in PAH-Ex vs. unexercised PAH with no difference between exercised and unexercised controls. Greater p-eNOS was measured in PAH-Ex lung compared to unexercised PAH, with no difference between exercised and unexercised controls. PAH-Ex also tended to have greater pulmonary eNOS than their unexercised counterparts. No greater exercise-induced CD45/68 infiltration was observed in RV of PAH compared to that of controls. In rats with moderate MCT-induced PAH, a single exercise bout does not increase acute RV inflammation but lowers pulmonary pressure, possibly mediated in part via pulmonary eNOS activation

Exercise Training Improves Cardiac and Skeletal Muscle Metabolism in Rats with Pulmonary Arterial Hypertension

poster abstractIn patients with pulmonary arterial hypertension (PAH), a shift from oxidative to glycolytic metabolism promotes right ventricular (RV) and skeletal muscle dysfunction that contributes to reduced exercise tolerance. As seen for other cardiopulmonary diseases, exercise training (ExT) may ameliorate this glycolytic switch in PAH and improve exercise capacity. The purpose of this research is to investigate ExT in a rat model of PAH on markers of glycolytic and oxidative metabolism in RV and skeletal muscle. Male Sprague-Dawley rats received monocrotaline (MCT, 40 mg/kg, s.q.) to induce PAH (n= 13), or saline, for healthy controls (n=5). After 2 wks, with MCT-induced PAH established, 6 wks of treadmill (TM) ExT was initiated for a subset of PAH animals (PAH-ExT, n= 6) and healthy controls (CON-ExT, n=3). ExT runs progressed up to 60 min at mild relative intensity, 50% of maximal aerobic capacity (VO2max). VO2max was assessed at baseline, in pre-training and post-training TM testing via analysis of expired gases. Abundance of Glut-1, a marker of glycolytic metabolism, was evaluated in cryosections of RV and soleus with immunofluorescent (IF) staining and quantification. Data are presented as mean±SE. MCT-ExT rats maintained aerobic capacity over 6 wks better than sedentary counterparts (MCT-SED)(VO2max= -134±109 vs. -521±129 ml/kg/hr, p=0.04) and was not different than CON-ExT (-201±31 ml/kg/hr, p=0.82). A lower abundance of Glut-1 was observed in both RV and soleus myocytes of PAH-ExT rats (MPI= 10.9 ±0.9 for RV; 13.7±0.8 for soleus) compared to PAH-SED rats (15.7±2.4, p=0.05, for RV; 17.4±1.4, p=0.04, for soleus) and was similar to CON-ExT rats (13.0±2.2, p=0.33, for RV; 9.0±2.3, p=0.26, for soleus), indicative of a shift toward greater dependency on oxidative metabolism. Exercise training attenuates functional decline following MCT administration in rats. Preservation of aerobic capacity may be explained by promotion of more efficient RV and skeletal muscle mitochondrial substrate utilization

Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo

<p>Abstract</p> <p>Background</p> <p>During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.</p> <p>Results</p> <p>In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the <it>de novo </it>expression of genes associated with extraembryonic differentiation.</p> <p>Conclusions</p> <p>These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted developmental potentials.</p

Embryonic stem cell-derived hemangioblasts remain epigenetically plastic and require PRC1 to prevent neural gene expression.

Many lineage-specific developmental regulator genes are transcriptionally primed in embryonic stem (ES) cells; RNA Pol(II) is bound at their promoters but is prevented from productive elongation by the activity of polycomb repressive complexes (PRC) 1 and 2. This epigenetically poised state is thought to enable ES cells to rapidly execute multiple differentiation programs and is recognized by a simultaneous enrichment for trimethylation of lysine 4 and trimethylation of lysine 27 of histone H3 (bivalent chromatin) across promoter regions. Here we show that the chromatin profile of this important cohort of genes is progressively modified as ES cells differentiate toward blood-forming precursors. Surprisingly however, neural specifying genes, such as Nkx2-2, Nkx2-9, and Sox1, remain bivalent and primed even in committed hemangioblasts, as conditional deletion of PRC1 results in overt and inappropriate expression of neural genes in hemangioblasts. These data reinforce the importance of PRC1 for normal hematopoietic differentiation and reveal an unexpected epigenetic plasticity of mesoderm-committed hemangioblasts

The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells

A panel of mutant embryonic stem (ES) cell lines lacking important chromatin modifiers was used to dissect the relationship between chromatin structure and replication timing, revealing the importance of several chromatin modifiers for maintaining correct replication of satellite sequences in pluripotent ES cells