Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming

SummaryMouse embryonic stem cells (mESCs) cultured under serum/LIF conditions exhibit heterogeneous expression of pluripotency-associated factors that can be overcome by two inhibitors (2i) of the MEK and GSK3 pathways. Several studies have shown that the “ground state” induced by 2i is characterized by global hypomethylation and specific transcriptional profiles, but little is known about the contributing effectors. Here we show that 2i conditions rapidly alter the global binding landscape of OCT4, SOX2, and NANOG. The dynamic binding influences enhancer activity and shows enrichment for regulators linked to Wnt and Erk signaling. Epigenomic characterization provided limited insights to the immediate transcriptional dynamics, suggesting that these are likely more secondary effects. Likewise, loss of the PRC2 component EED to prevent H3K27me3 deposition had minimal effect on the transcriptome, implying that it is largely dispensable for continued repression of bivalent genes and de novo silencing in 2i

ATTENUATION OF IRON OVERLOAD AND EFFECT OF ANTIOXIDANTS SUPPLEMENTATION ON OXIDATIVE STRESS IN HOMOZYGOUS β-THALASSEMIA

It has been projected that ferritin and iron yoke in homozygous thalassemic children is coupled with the enhanced free radical formation and blemished in antioxidative defense coordination. Aim: The purpose of the current study was to analyze the consequence of serum iron, erythrocyte catalase, and erythrocyte superoxide dismutase (ESOD) in patients with beta-thalassemia major. Method: 60 beta-thalassemia major patients were studied before and after supplementation of A – Z antioxidants for 20 weeks, and status were compared with 60 age and sex-matched healthy normal.    Serum Iron estimation was carried out by Ramsay’s Dipyridyl Method. Estimation of erythrocyte superoxide dismutase was done by Kajari Das Method. The levels of Catalase concentration in erythrocytes were analyzed by the Goth method.  All the objectives mentioned above were run by using a UV visible Spectrophotometer (Systronix). Results: A marked enhancement was seen in the intensity of serum iron, and superoxide dismutase (p<0.001) with parallel decline was observed in the level of erythrocyte catalase (p<0.001) in homozygous thalassemia patients when compared with healthy subjects. After 20 weeks of regular supplementation of antioxidants A-Z syrup, which consists of multimineral multivitamins, the concentration of catalase was increased whereas iron and ESOD (p<0.001) were reduced significantly when compared with normal and baselines thalassemic patients. Conclusion: Due to multiple blood transfusions, beta-thalassemia major children are at advanced risk due to secondary iron surplus and intense oxidative stress. Such kind of circumstances may be handled with supplementation of antioxidants A-Z syrup with their regular treatment. &nbsp

ATTENUATION OF IRON OVERLOAD AND EFFECT OF ANTIOXIDANTS SUPPLEMENTATION ON OXIDATIVE STRESS IN HOMOZYGOUS β-THALASSEMIA

It has been projected that ferritin and iron yoke in homozygous thalassemic children is coupled with the enhanced free radical formation and blemished in antioxidative defense coordination. Aim: The purpose of the current study was to analyze the consequence of serum iron, erythrocyte catalase, and erythrocyte superoxide dismutase (ESOD) in patients with beta-thalassemia major. Method: 60 beta-thalassemia major patients were studied before and after supplementation of A – Z antioxidants for 20 weeks, and status were compared with 60 age and sex-matched healthy normal.    Serum Iron estimation was carried out by Ramsay’s Dipyridyl Method. Estimation of erythrocyte superoxide dismutase was done by Kajari Das Method. The levels of Catalase concentration in erythrocytes were analyzed by the Goth method.  All the objectives mentioned above were run by using a UV visible Spectrophotometer (Systronix). Results: A marked enhancement was seen in the intensity of serum iron, and superoxide dismutase (p<0.001) with parallel decline was observed in the level of erythrocyte catalase (p<0.001) in homozygous thalassemia patients when compared with healthy subjects. After 20 weeks of regular supplementation of antioxidants A-Z syrup, which consists of multimineral multivitamins, the concentration of catalase was increased whereas iron and ESOD (p<0.001) were reduced significantly when compared with normal and baselines thalassemic patients. Conclusion: Due to multiple blood transfusions, beta-thalassemia major children are at advanced risk due to secondary iron surplus and intense oxidative stress. Such kind of circumstances may be handled with supplementation of antioxidants A-Z syrup with their regular treatment. &nbsp

ALTERATION IN SERUM ZINC AND COPPER CONCENTRATIONS AND EFFECT OF ORAL THERAPEUTIC SUPPLEMENTATION OF ZINC ON TRANSFUSION DEPENDANT BETA THALASSEMIA MAJOR PATIENTS

Zinc is one of the essential micronutrients in human and act as a cofactor for more than 300 enzymes and plays an essential role in human growth and development. It has been observed that there was low serum zinc and elevated  copper level in β-thalassemia major compared with normal. Zinc deficiency is considered one of the main factors contributing to growth, cardiovascular diseases, and puberty disorders in β-thalassemic patients. Aim: The goal of the study was to scrutinize the impact of serum zinc and copper concentration in patients with beta-thalassemia major and also to observe the effect of zinc supplementation on transfusion dependent beta-thalassemia patients for six months. Method: 52 beta-thalassemia major patients were studied before and after supplementation of zinc for six months, and status was compared with 52 age and sex-matched healthy normal.  Serum zinc and copper concentration were measured by atomic absorption spectrophotometry (AAS) method. Result: There was a significant depleted activity of  serum zinc level (p<0.001), and the copper level was increased significantly (p<0.001) in patients when compared with normal. After six months of supplementation of zinc, there was a significantly enhanced zinc concentration (p<0.001),and copper was marginally increased (p>0.05) when compared with normal and baselines. Conclusion: Beta Thalassemia  major children are on numerous blood transfusions all the way through their life. Due  to this  thalassemic children are at risk of secondary iron burden. This further leads to the  enhanced  oxidative stress. One of the way to may overcome this situation to supply regular zinc supplementation along with treatment, which may be helpful to manage the situation. &nbsp

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities

DNA methylation dynamics of the human preimplantation embryo

In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell type-specific regulation directed by DNA binding factors1-3. This comparatively static landscape dramatically contrasts the events of fertilization, where the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, though methylation is maintained at several notable features4-7. While these dynamics have been extensively characterized in the mouse, only limited observations are available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved8-10. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell (ESC) derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of persistent maintenance are primarily restricted to gene bodies. While most features share similar dynamics to mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island (CGI) promoters that extend beyond known Imprint Control Regions (ICRs). Retrotransposon regulation is also highly diverse and transitions from maternally to embryonically expressed, species-specific elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation

PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells

Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Elsevier. The published article can be found at: http://www.cell.com/cell-reports/hom

Identification of Predictive Cis-Regulatory Elements Using a Discriminative Objective Function and a Dynamic Search Space

<div><p>The generation of genomic binding or accessibility data from massively parallel sequencing technologies such as ChIP-seq and DNase-seq continues to accelerate. Yet state-of-the-art computational approaches for the identification of DNA binding motifs often yield motifs of weak predictive power. Here we present a novel computational algorithm called MotifSpec, designed to find predictive motifs, in contrast to over-represented sequence elements. The key distinguishing feature of this algorithm is that it uses a dynamic search space and a learned threshold to find discriminative motifs in combination with the modeling of motifs using a full PWM (position weight matrix) rather than <i>k</i>-mer words or regular expressions. We demonstrate that our approach finds motifs corresponding to known binding specificities in several mammalian ChIP-seq datasets, and that our PWMs classify the ChIP-seq signals with accuracy comparable to, or marginally better than motifs from the best existing algorithms. In other datasets, our algorithm identifies novel motifs where other methods fail. Finally, we apply this algorithm to detect motifs from expression datasets in <i>C</i>. <i>elegans</i> using a dynamic expression similarity metric rather than fixed expression clusters, and find novel predictive motifs.</p></div

modENCODE ChIP-seq results.

<p>Binding specificities for four <i>C</i>. <i>elegans</i> transcription factors as learnt from ChIP-seq data from the modENCODE project.</p