Myst4 is required for neural differentiation of mouse embryonic stem cells

MARÍA SOLEDAD COSENTINO; ARIEL WAISMAN; CAMILA VÁZQUEZ ECHEGARAY; CLAUDIA SOLARI; MARCOS FRANCIA; SANTIAGO MIRIUKA; LINO BARAÑAO; ALEJANDRA GUBERMAN

Congreso; LXI Reunión de la Sociedad Argentina de Investigación Clínica; 2016

The transition between transcriptional programs associated with Stem Cell (SC) differentiation is related to changes in chromatin structure. In this work we studied Myst4, a transcriptional coactivator with histone acetyltransferase activity. It was reported that Myst4 gene has a regulatory element highly occupied by Embryonic SC (ESC) key transcription factors (TFs), but the relevance of this protein in ESC remains to be established. Additionally, this gene is important for the establishment and self-renewal of adult neural SC, and loss of only one allele in humans leads to intellectual disability. We have previously shown that Myst4 is expressed in mouse ESC (mESC) and is repressed during differentiation. Moreover, our previous results also suggest that its expression is regulated by pluripotency TFs. To study the role of Myst4 in the maintenance of ESC?s properties, we used the CRISPR/Cas9 strategy to generate mESC knock-out cell lines. Most of the clones analyzed presented indel mutations. We selected a clone with a frameshift mutation that generated a premature stop codon in both alelles (M4 -/-), and confirmed the lack of Myst4 protein expression by Western blot. This clone displayed normal morphology and had no significant differences to the wild type (WT) control cells regarding pluripotency markers expression. Surprisingly, M4-/- mESC failed to differentiate to neural derivatives during a directed differentiation protocol, with most of the cells dying at day 12. Gene expression analysis during initial stages of neural differentiation showed that M4 -/- mESC had lower expression of neural progenitor and neuron markers than WT cells. These results suggest that Myst4 is required for the differentiation of mESC to neurons, and provides a platform to study the epigenetic mechanisms of normal development as well as human disease. We consider that understanding the processes involved in ESC chromatin structure regulation is critical to their future application.