Modular affinity-labeling of the cytosine demethylation base elements in DNA

Abstract5-methylcytosine is the most studied DNA epigenetic modification, having been linked to diverse biological processes and disease states. The elucidation of cytosine demethylation has drawn added attention the three additional intermediate modifications involved in that pathway—5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine—each of which may have distinct biological roles. Here, we extend a modular method for labeling base modifications in DNA to recognize all four bases involved in demethylation. We demonstrate both differential insertion of a single affinity tag (biotin) at the precise position of target elements and subsequent repair of the nicked phosphate backbone that remains following the procedure. The approach enables affinity isolation and downstream analyses without inducing widespread damage to the DNA.

Abstract 17245: Deficiency of Tet Methylcytosine Dioxygenase 2 (tet2),A Key Enzyme in Cytosine Demethylation,Is an Epigenetic Driver of Inflammation and Disease Progression in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by remodelling of distal pulmonary arteries associated with inflammation, endothelial cells (EC) dysfunction and a pro-proliferative/anti-apoptotic phenotype in pulmonary arterial smooth muscle cells (PASMC). Tet methylcytosine dioxygenase 2 (Tet2) is a key enzyme in cytosine demethylation that is crucial for epigenetic control of gene expression. Deficiency of Tet2 expression in myeloid cellsresults in increased inflammatory cytokine levels. Moreover, impaired Tet2 expression in EC has also been associated with decreased autophagy, as well as EC dysfunction. In addition, Tet2 silencing contributes to pro-proliferative phenotype of SMC. Thus, we hypothesized that Tet2 deletion would contribute to PAH by upregulation of inflammation and induction of PASMC proliferation and EC dysfunction. Methods and Results: To assess the ability of Tet2 deficiency to induce PAH we assessed cardiopulmonary hemodynamics in a conditional hematopoietic and endothelial cells Tet2 -/- mouse model. Tet2 -/- mice developed PAH associated with significant increases of right ventricular systolic pressure (RVSP), elevation of total pulmonary resistance (TPR) and increased vascular wall thickness of distal pulmonary arteries. Tet2 -/- lung tissue as well as macrophages and EC sorted from the lung displayed aberrant inflammatory cytokine signalling with robust overexpression of IL1βnoted on a Nanostring immune gene expression panel. In vitro IL1βincreased PASMC proliferation measured using EDU. Downregulation of TET2 in PASMC using siRNA resulted in a similar PAH-like phenotype. Moreover, lung from Tet2 -/- mice exhibited an alteration of vasoactive mediator gene expression (increased endothelin-1 (ET1), Arginase 2 (Arg2) and decreased eNOS). Finally we showed that downregulation of Tet2 (achieved by nebulization of si-Tet2) in rats with monocrotaline-induced PAH exacerbated disease severity (increasing TPR adverse pulmonary vascular remodelling).This phenotype was independent of the sex, was associated with a moderate decrease in RV function but had no significant effect on LV hemodynamic parameters. Conclusions: Tet2 is a protective epigenetic regulator that when conditionally downregulated in hematopoietic and endothelial cells leads toPAH by inducing a state of inflammation that contributes to PASMC proliferation and EC dysfunction which drives adverse pulmonary vascular remodelling.

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation.

127. EPIGENETIC REPROGRAMMING IN ZYGOTES INVOLVES THE GLOBAL CYTOSINE DEMETHYLATION OF BOTH THE PATERNAL AND MATERNAL GENOMES

Epigenetic reprogramming is essential for normal development and has been held to occur in a different manner for the paternally and maternally inherited genomes. The current paradigm implicates active global demethylation of the paternal pronucleus soon after fertlization, but passive demethylation of maternally-derived genome over many cell-cycles. This parent-of-origin difference has been difficult to reconcile with other biological processes prompting us to re-examine this evidence. DNA methylation levels were examined in mouse zygotes by immunolocalization with methylcytosine specific antibodies. Zygotes were isolated from the oviduct at times after hCG and staged for pronuclei maturity (PN1-5, least to most mature) or metaphase commencement. We found methylation levels to be high in PN1-2 stage pronuclei but then progressively declined. By PN5 stage methylcytosine staining was greatly diminished. Yet, contrary to the current paradigm, demethylation generally occurred in both the male and female pronucleus. We found no methylcytosine staining in any metaphase chromosomes. The contrast of our results with those widely cited prompted us to review the methodology previously used. In previous studies zygotes that had been collected after fertilization and then cultured in vitro, or produced by IVF and then cultured were used. When we prepared zygotes by these methods we found that many PN5-stage cultured zygotes displayed relatively more demethylation of the male pronucleus than the female. When zygotes were generated by IVF this asynchrony was further exacerbated. In contrast to the zygotes collected directly from the reproductive tract, metaphase chromosomes in cultured post-syngamal zygotes commonly showed extensive methylcytosine staining. Our results show that the normal process of epigenetic reprogramming in the mouse involves global demethylation of both the paternal and maternal genomes. This was variably perturbed (particularly in the female pronucleus) by IVF and zygote culture.