Histone methyltransferase DOT1L activity is a critical and stage specific instructor for neuronal differentiation of embryonic stem cells

VILLARREAL A; VOGEL T

International Society for Developmental Neuroscience

Congreso; 21st Biennial Meeting of the International Society for Developmental Neuroscience; 2016

DOT1L methylates Histone 3 at K79 having important functions in cell cycle regulation and DNA repair. Increased H3K79me2 mostly correlates to active transcription, however further evidence also show opposite results. Our group reported that the transcription factor AF9 regulates correct layering of brain cortex by recruiting DOT1L at the Tbr1 gene and repressing its transcription. Further impairment of DOT1L activity leads to an increase in cortical cell precursor death by activating the unfolded protein pathway triggered by ATF4/DDIT3. We now aim to understand the precise role of DOT1L during neuronal differentiation using genome wide analyses. We differentiated embryonic stem cells (mESC) using a model that allows generation of cortical neuron cultures by going throw a stage of radial glia like cells recapitulating the process observed in vivo. We analyze DOT1L expression and activity during this process and performed pharmacological inhibition of DOT1L at different time points analyzing its influence on neuronal differentiation.We used a transgenic mESC line that endogenously expresses DOT1L fused to HA/FLAG. The differentiation process was validated by assessing correct temporal expression of specific markers (OCT4/PAX6/NESTIN/MAP2/TUJ1) by immunofluorescence and immunoblots. The cell morphology for each time point was also addressed by immunofluorescence. Immunoblot analysis showed an increase in H3K79me2 in differentiated neurons when compared to mESC and early stages of neural differentiation. However, qPCR results showed constant levels of DOT1L expression. Importantly, we observed that neural progenitor cells are sensitive to DOT1L pharmacological inhibition in a specific phase of the differentiation process. Such sensitivity was displayed by decreased numbers of HuC/D(+) neurons. In contrast, mESC proliferation and survival were not affected.Our data reflects that DOT1L activity is required at specific time points during neuronal differentiation. Ongoing experiments will elucidate which cell processes are specifically affected and which chromatin domains become enriched with H3K79me2.