The type of DNA damage response after decitabine treatment depends on the level of DNMT activity.

Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

Redirected nuclear glutamate dehydrogenase supplies Tet3 with α-ketoglutarate in neurons.

Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.

ECE2 regulates neurogenesis and neuronal migration during human cortical development.

During embryonic development, excitatory projection neurons migrate in the cerebral cortex giving rise to organised layers. Periventricular heterotopia (PH) is a group of aetiologically heterogeneous disorders in which a subpopulation of newborn projection neurons fails to initiate their radial migration to the cortex, ultimately resulting in bands or nodules of grey matter lining the lateral ventricles. Although a number of genes have been implicated in its cause, currently they only satisfactorily explain the pathogenesis of the condition for 50% of patients. Novel gene discovery is complicated by the extreme genetic heterogeneity recently described to underlie its cause. Here, we study the neurodevelopmental role of endothelin-converting enzyme-2 (ECE2) for which two biallelic variants have been identified in two separate patients with PH. Our results show that manipulation of ECE2 levels in human cerebral organoids and in the developing mouse cortex leads to ectopic localisation of neural progenitors and neurons. We uncover the role of ECE2 in neurogenesis, and mechanistically, we identify its involvement in the generation and secretion of extracellular matrix proteins in addition to cytoskeleton and adhesion.