Biology subjects

Traube, F. R.

Publications and source records attributed to Traube, F. R..

8 recordsLinked to original sources

bioRxiv2025

Increasing TET expression and 5-hydroxymethylcytosine formation by a carbocyclic 5-aza-2'-deoxy-cytidine antimetabolite

Ten-eleven translocation (TET) enzymes are critical epigenetic regulators, which oxidize the methylated cytosine nucleobase 5-methyl-dC (5mdC) in the genome to 5-hydroxymethyl-dC (5hmdC) in an -ketoglutarate-dependent manner. Because the presence of 5mdC in the promoter region of a given gene silences its expression, this oxidation goes in hand with the reactivation of such silenced genes. In different highly aggressive cancers such as acute myeloid leukemia (AML) and glioblastoma, loss of TET enzyme function and therefore reduced 5hmdC levels pave the way for tumor development. Impairment of TET activity can occur through metabolic inhibition, through loss-of-function mutations in TET genes themselves, and finally through suppression of TET-expression via epigenetic silencing. Reactivation of TET enzyme expression represents a major aim of epigenetic cancer therapy. Here we show that the carbocyclic antimetabolite 5-aza-2deoxycytidine (cAzadC), which is supposed to suppress the methylation of DNA during replication, leads to a substantial increase of TET2 expression and strongly increasing 5hmdC levels. We show that the treatment with cAzadC goes in hand with the broad reactivation of the cellular anti-tumor responses. With patient-derived xenograft AML-mouse models, we show that this translates into a strongly improved anti-cancer effect in vivo.

cancer biology↗

bioRxiv2025

Crosstalk between the Methyl-Cytosine Dioxygenase TET3 and the Methyl-CpG-binding protein MECP2 Controls Neuronal Maturation

Active DNA demethylation depends on Ten-Eleven-Translocation (TET) enzymes, which oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) and further derivatives. Mutations in TET3, encoding the predominant neuronal isoform, lead to Beck-Fahrner syndrome, a neurodevelopmental disorder. Using human iPSC-derived neurons, we show that TET3 is dispensable for neuronal specification but critical for subsequent maturation. Differentiating TET3-deficient neurons exhibit delayed transcriptional and proteomic transitions, altered synaptic signatures, and impaired network activity, indicating delayed functional maturation. Mechanistically, we identified an interaction between TET3 and the mC/hmC-binding protein MECP2, pathogenic variants of which cause Rett syndrome. MECP2 negatively regulates TET3 activity, as demonstrated in functional assays and by inverse hmC patterns in MECP2- and TET3-deficient neurons. Despite this, MECP2- and TET3-deficient neurons exhibit highly similar phenotypes later in differentiation. Our findings uncover a functional interplay between TET3 and MECP2 that coordinates DNA methylation and chromatin dynamics during neuronal maturation, suggesting a shared pathogenic mechanism in Beck-Fahrner and Rett syndromes.

neuroscience↗

bioRxiv2024

CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. MotivationThe CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing1-8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS)9-12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.

biochemistry↗

bioRxiv2023

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 applied against myeloid cancers, important aspects of their mode of action remain unknown, which highly limits 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, contradicting an RNA-dependent effect of Azacytidine. By applying spatial proteomics, we show that Decitabine induces a strictly DNMT-dependent multifaceted DNA- damage response based on chromatin-recruitment of various repair-associated proteins. The choice of DNA-repair pathway herby depends on the severity of Decitabine-induced DNA- lesions. While mismatch (MMR) and base-excision DNA repair (BER) as well as RAD50- dependent DNA double-strand break repair are always activated in response to Decitabine, Fanconi anemia-dependent DNA-repair combined with homologous recombination is only activated when DNMT-activity is moderate. In contrast, high DNMT-activity and therefore immense replication stress, induce DNA repair by non-homologous and alternative end-joining.

biochemistry↗

bioRxiv2022

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2-deoxycytidine by methylating 2-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity towards AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response towards AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

molecular biology↗

bioRxiv2022

Epigenetic anti-cancer treatment with a stabilized carbocyclic Decitabine analogue

5-Aza-2-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

cancer biology↗

bioRxiv2018

TET1 drives global DNA demethylation via DPPA3-mediated inhibition of maintenance methylation

Genome-wide DNA demethylation is a unique feature of mammalian development and naive pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naive pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.

developmental biology↗

bioRxiv2018

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

The TET-oxidized cytosine derivatives, 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), are considered DNA demethylation intermediates as well as stable epigenetic marks in mammals. We compared modified cytosine and enzyme levels in TET-knockout cells during naive pluripotency exit and found distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation. The divergent modified cytosine levels argue for independent consecutive oxidation steps in vivo with broad implications for epigenetic regulation.

cell biology↗