DOT1L methyltransferase interacts with NPM1 outside the nucleolus: the study of a possible non-histone target of DOT1L.

VILLARREAL A; HELLBACH N; HEIDRICH S; VOGEL T

Barcelona

Congreso; Coding and Non-Coding Functions of the Genome. 2015 BCEC.; 2015

International center for scientific debate

DOT1L (disruptor of telomeric silencing 1-like) also known as KMT4, is the methyltransferase responsible for the mono, di and trimethylation of histone 3 at the lysine 79 with important functions in cell cycle regulation, DNA repair and probably in cell differentiation during development. It has been reported that DOT1l might interact with other non-histone proteins such as Nucleophosmin (NPM1), a fact that would broaden DOT1L functions beyond histone methylation. Furthermore, both proteins have shown to have important functions in leukemia.Our aim in this project is to analyze the interaction between DOT1l and NPM1 within the cell and to reveal the purpose of this protein complex.To validate DOT1l/NPM1 interaction and localization of the complex, Neuro2A cells were transfected with a DOT1l_HA_FLAG overexpression vector and processed for immunoprecipitation, immunofluorescence and proximity ligation assays. Proliferation was assessed by measuring BrdU incorporation and NPM1 expression and localization was analyzed using qPCR, western blot and confocal imaging. Loss of function experiments were performed using a shRNA for DOT1l knock down or the inhibitor SGC0946.So far our data indicates that the DOT1l/NPM1 complex localizes outside the nucleolus. We observed no changes in NPM1 expression, localization or impairments in cell proliferation upon DOT1l-KD or inhibition. Stable NPM1 expression was also confirmed in cortical neurons, fibroblasts, and in a leukemia cell line.Since NPM1 relocalizes outside the nucleolus, not only during mitosis but also during DNA damage repair, further experiments using DNA damaging agents will be conducted to elucidate the functionality of the NPM1/DOT1l complex.