DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.

Metastatic methicillin-resistant Staphylococcus aureus infection with infective endocarditis, pulmonary septic emboli, axillary abscess with a subacute presentation in a patient with chronic end-stage kidney disease on maintenance haemodialysis.

A male adult patient on maintenance haemodialysis due to end-stage diabetic nephropathy presented with low-grade intermittent fever, cough and generalised weakness for 3 weeks. Initial blood investigations revealed an elevated neutrophil count with raised inflammatory markers. Chest CT revealed loculated hydropneumothorax with multiple cavitary nodules. Repeated blood cultures from the cuffed tunnelled catheter site and the right arm and sputum cultures were negative for pyogenic bacteria and yeast aetiology. The patient complained about left axillary pain on the fourth day of admission. Ultrasound-guided percutaneous aspiration from an axillary focal collection and subsequent culture revealed a methicillin-resistant Staphylococcus aureus (MRSA) infection. Echocardiography detected multiple vegetations on the tricuspid valve. The patient responded clinically to vancomycin and removal of the permanent catheter. This was a case of a tunnelled catheter-related metastatic MRSA infection with infective endocarditis, pulmonary septic embolism with a subacute presentation, and repeated blood culture negativity.

Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells.

Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.

Effect of Disease-Associated Germline Mutations on Structure Function Relationship of DNA Methyltransferases.

Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the activity of mammalian DNA methyltransferases (DNMTs) are not fully understood. Recent advances in whole genome association studies have helped identify mutations and genetic alterations of DNMTs in various diseases that have a potential to affect the biological function and activity of these enzymes. Several of these mutations are germline-transmitted and associated with a number of hereditary disorders, which are potentially caused by aberrant DNA methylation patterns in the regulatory compartments of the genome. These hereditary disorders usually cause neurological dysfunction, growth defects, and inherited cancers. Biochemical and biological characterization of DNMT variants can reveal the molecular mechanism of these enzymes and give insights on their specific functions. In this review, we introduce roles and regulation of DNA methylation and DNMTs. We discuss DNMT mutations that are associated with rare diseases, the characterized effects of these mutations on enzyme activity and provide insights on their potential effects based on the known crystal structure of these proteins.

Xeno-Free Cryopreservation of Bone Marrow-Derived Multipotent Stromal Cells from Callithrix jacchus.

In the previous decade, numerous biobanks were established and have created large markets for the storage of bioactive compounds, cells, and tissues for medical and diagnostic applications. For in vivo clinical and therapeutic purposes, it is critical to use well-defined and xeno-free components during cultivation, preservation, and transplantation of biological material. Safe and efficacious storage of bioactive molecules, cells, and tissues, without the addition of undefined medium components, minimizes risks of zoonotic disease transmission and is thus an essential and desirable prerequisite for biobanks. This gives rise to a need for well-characterized and serum-free freezing media for application in cryopreservation. For this purpose, cryobiological additives such as methylcellulose, poloxamer-188, and α-tocopherol, which have previously been shown to exhibit a cytoprotective activity, have been investigated for cryoprotection on stem cells. With this strategy, the application of fetal bovine serum (FBS) could be avoided and the concentration of toxic cryoprotective agents such as dimethyl sulfoxide (DMSO) could be reduced. Our results suggest that the viability, as well as the adipogenic and osteogenic differentiation capacity of the thawed bone marrow-derived multipotent stromal stem cells, could be maintained using a freezing medium without FBS consisting of methylcellulose, poloxamer, and α-tocopherol with only 2.5% DMSO (% v/v).

Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation.

Establishment, characterization, and differentiation of a karyotypically normal human embryonic stem cell line from a trisomy-affected embryo.

Derivation of human embryonic stem cell (hESC) lines from chromosomally or genetically abnormal embryos obtained following preimplantation genetic diagnosis (PGD) is of immense interest to study various kinds of genetic disorders. In this study, we have established a new hESC line Relicell(®)hES4, isolated from an aneuploid embryo. Derivation of this cell line was achieved by isolation of the inner cell mass (ICM) by mechanical method. Karyotype analysis showed that the hESC line is euploid having 46 chromosomes, contrary to our expectations. The undifferentiated cells exhibited long-term proliferation capacity and expressed markers typical for hESC, such as OCT4, NANOG, and SSEA4. A comparative microarray study was carried out to analyze the transcription profile of Relicell(®)hES4 along with three other normal hESC line generated earlier in our lab. Relicell(®)hES4 manifested pluripotent differentiation potential both in vivo and in vitro. The cells were also induced to form neurons, cardiomyocytes, and pancreatic ß islets. The generation of a normal hESC line from an abnormal embryo points to the fact that even such embryos can be considered for deriving new hESC lines instead of discarding them. The data represented here are the first detailed report on characterization and differentiation of an Indian hESC line generated from a PGD analyzed embryo.