2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

peer reviewed2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation

peer reviewedMidbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity

Potential role of different DNA-glycosylases in the direct deformylation of 5-formylcytosine.

The regulation of transcription results from a complex interplay of molecular factors and markers that co-operate during all the stages of the gene expression. The instruction for the transcriptional expression is not only stored in the four canonical bases fashion but in the so-called epigenetic hallmarks, heritable modifications and chromatin rearrangements that are able to regulate the genome accessibility. 5-methyl-deoxycytidine(mdC) is an epigenetic modification able to mark a gene or an entire chromosome for the transcriptional silencing, therefore its deposition and removal is finely regulated by the cell. An active demethylation pathway has been demonstrated to occur through an oxidation cascade of mdC performed by the TET enzymes, leading to the formation of 5-hydroxymethyl-deoxycytidine(hmdC), 5-formyl-deoxycytidine(fdC) and 5-carboxy-deoxycytidine(cadC). The enzymes of the Base excision repair (BER) mechanism then catalyse the removal of the oxidised base, restoring the deoxycytidine(dC). The DNA-glycosylases are the first actors of this pathway as they recognize the modified base and cleave the N-glycosidic bond leaving an apyrimidinic site (AP). Nevertheless, this pathway creates single or double strand breaks which result detrimental for the genome stability. Different models have thus been proposed as alternative unharmful mechanisms for the mdC active removal. This project aims to investigate the role of different DNA-glycosylases as potential catalysts of the direct deformylation of fdC. To this extent, the deformylation assay was performed incubating the DNA-glycosylase in presence of an oligonucleotide carrying a 2’-Fluorinated-formyl-deoxycytidine(F-fdC). This modified base is fluorinated at the 2’ of the ribose ring thus it sterically impedes the DNA-glycosylase access to the N-glycosidic bond. The results were finally investigated using mass spectrometry analysis