Gawronski, M.; Starczak, M.; Wasilow, A.; Dziaman, T.; Olinski, R.; Gackowski, D.

Background: The TET family of proteins - TET1, TET2, and TET3 - are -KG and Fe2+ dependent dioxygenases that play crucial roles in active DNA demethylation and the deposition of epigenetic marks such as 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. TET proteins can also oxidize thymine to 5-hydroxymethyl uracil - a modification whose role is still poorly understood. TET proteins add a new layer of information in regulating gene expression, cellular development, and lineage specification. Dysregulation of TET activity is implicated in various cancers, especially in hematological malignancies, where TET2 loss-of-function mutations are prevalent. TET2's role in hematopoiesis is critical, as its knockdown skews progenitor differentiation toward the myeloid lineage and drives carcinogenesis. Therefore, restoring the lost activity of TET proteins is often proposed as an important component of cancer treatment. This study explores the distinct contributions of TET paralogs in generating active demethylation products in malignant cells. It examines whether vitamin C, a known cofactor of many dioxygenases, can compensate for the loss of specific TET paralogs. We applied a highly sensitive and specific methodology (2D-UPLC-MS/MS) to assess TET activity in the HAP1 cell line with single and double TET functional knockouts and in cells with the activity of all TET proteins impaired. Results: Our findings reveal that TET2 is essential for all steps of iterative oxidation, and its loss has the most significant effect on 5-hydroxymethylcytosine and 5-formylcytosine levels. Vitamin C enhances TET activity and increases the levels of these oxidation products. However, its effect in TET2 knockout cells is limited; Vitamin C increased cytosine modification levels in TET2KO cells, but not to the extent observed in treated wild-type cells, indicating incomplete compensation for TET2 loss. Conclusions: Our results demonstrated that each TET protein has a distinct, separate contribution to generating active demethylation products. The absence of individual TET paralog is linked with the specific pattern of active demethylation products in DNA, which is preserved after vitamin C treatment. Therefore, the deletion of one of the TET enzymes cannot be compensated for by the increased activity of the other TET family members, highlighting the unique roles of each TET paralog in epigenetic regulation.