DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to ...5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown.
Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters.
We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.
Pharmacologic concentrations of ascorbate may be effective in cancer therapeutics. We hypothesized that ascorbate concentrations achievable with i.v. dosing would be cytotoxic in pancreatic cancer ...for which the 5-year survival is <3%.
Pancreatic cancer cell lines were treated with ascorbate (0, 5, or 10 mmol/L) for 1 hour, then viability and clonogenic survival were determined. Pancreatic tumor cells were delivered s.c. into the flank region of nude mice and allowed to grow at which time they were randomized to receive either ascorbate (4 g/kg) or osmotically equivalent saline (1 mol/L) i.p. for 2 weeks.
There was a time- and dose-dependent increase in measured H(2)O(2) production with increased concentrations of ascorbate. Ascorbate decreased viability in all pancreatic cancer cell lines but had no effect on an immortalized pancreatic ductal epithelial cell line. Ascorbate decreased clonogenic survival of the pancreatic cancer cell lines, which was reversed by treatment of cells with scavengers of H(2)O(2). Treatment with ascorbate induced a caspase-independent cell death that was associated with autophagy. In vivo, treatment with ascorbate inhibited tumor growth and prolonged survival.
These results show that pharmacologic doses of ascorbate, easily achievable in humans, may have potential for therapy in pancreatic cancer.
It is well established that environmental exposures can modify the profile of heritable factors in an individual's germ cells, ultimately affecting the inheritance of phenotypes in descendants. ...Similar to exposures, an ancestor's genotype can also affect the inheritance of phenotypes across generations, sometimes in offspring who do not inherit the genetic aberration. This can occur via a variety of prenatal, in utero, or postnatal mechanisms. In this review, we discuss the evidence for this process in mammals, with a focus on examples that are potentially mediated through the germline, while also considering alternate routes of inheritance. Noninherited ancestral genotypes may influence descendant's disease risk to a much greater extent than currently appreciated, and focused evaluation of this phenomenon may reveal novel mechanisms of inheritance. Summary sentence This is a review of the evidence that non-inherited ancestral alleles can influence the phenotypes of descendants via a variety of mechanisms including germline, in utero, and postnatal processes. Graphical Abstract
Unplanned extubations (UEs) can be a frequent problem and are associated with adverse outcomes. This quality improvement initiative sought to reduce UEs from tube dislodgement in a level IV NICU ...utilizing methods applicable to other ICUs and performed with minimal monetary funds.
From January 2019 to July 2023, an interdisciplinary quality improvement team used the Model for Improvement and performed sequential interventions to improve the outcome measure of UEs per 100 ventilator days. Process measures included adherence to a modified, site-specific UE care bundle derived from the Solutions for Patient Safety network, whereas the number of endotracheal tube-related pressure injuries was used as a balancing measure. Statistical process control charts and established rules for special cause variation were applied to analyze data.
Sequential interventions reduced the rate of UEs from a baseline of 2.3 to 0.6 UEs per 100 ventilator days. Greater than 90% adherence with the UE care bundle and apparent cause analysis form completion occurred since December 2020. There were no endotracheal tube-related pressure injuries.
A sustained reduction in UEs was demonstrated. Leveraging a multidisciplinary team allowed for continuous UE analysis, which promoted tailored consecutive interventions. UE care bundle audits and the creation of a postevent debrief guide, which helped providers share a common language, were the most impactful interventions. Next steps include disseminating these interventions to other ICUs across our hospital enterprise. These low-cost interventions can be scalable to other NICUs and PICUs.
Hematopoietic development and homeostasis are based on hematopoietic stem cells (HSCs), a pool of ancestor cells characterized by the unique combination of self-renewal and multilineage potential. ...These two opposing forces are finely orchestrated by several regulatory mechanisms, comprising both extrinsic and intrinsic factors. Over the past decades, several studies have contributed to dissect the key role of niche factors, signaling transduction pathways, and transcription factors in HSC development and maintenance. Accumulating evidence, however, suggests that a higher level of intrinsic regulation exists; epigenetic marks, by controlling chromatin accessibility, directly shape HSC developmental cascades, including their emergence during embryonic development, maintenance of self-renewal, lineage commitment, and aging. In addition, aberrant epigenetic marks have been found in several hematological malignancies, consistent with clinical findings that mutations targeting epigenetic regulators promote leukemogenesis. In this review, we will focus on both normal and malignant hematopoiesis, covering recent findings that illuminate the epigenetic life of HSCs.
Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of ...mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.
Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis ...and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.
Hippocampal-dependent deficits in learning and memory formation are a prominent feature of traumatic brain injury (TBI); however, the role of the hippocampus in cognitive dysfunction after concussion ...(mild TBI) is unknown. We therefore investigated functional and structural changes in the swine hippocampus following TBI using a model of head rotational acceleration that closely replicates the biomechanics and neuropathology of closed-head TBI in humans. We examined neurophysiological changes using a novel ex vivo hippocampal slice paradigm with extracellular stimulation and recording in the dentate gyrus and CA1 occurring at 7 days following non-impact inertial TBI in swine. Hippocampal neurophysiology post-injury revealed reduced axonal function, synaptic dysfunction, and regional hyperexcitability at one week following even "mild" injury levels. Moreover, these neurophysiological changes occurred in the apparent absence of intra-hippocampal neuronal or axonal degeneration. Input-output curves demonstrated an elevated excitatory post-synaptic potential (EPSP) output for a given fiber volley input in injured versus sham animals, suggesting a form of homeostatic plasticity that manifested as a compensatory response to decreased axonal function in post-synaptic regions. These data indicate that closed-head rotational acceleration-induced TBI, the common cause of concussion in humans, may induce significant alterations in hippocampal circuitry function that have not resolved at 7 days post-injury. This circuitry dysfunction may underlie some of the post-concussion symptomatology associated with the hippocampus, such as post-traumatic amnesia and ongoing cognitive deficits.
PDF
