Cortical neurogenesis depends on the balance between self‐renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single‐cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self‐renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Cortical neurogenesis depends on the tight balance between self-renewal and differentiation of apical progenitors (APs), the key progenitor type generating all other neural cells including neocortical neurons. We here report the activity of the histone methyltransferase DOT1L as a gatekeeper for AP cell identity. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we explore consequences of DOT1L inhibition on AP lineage progression during neurogenesis in the embryonic mouse neocortex. At the cellular level, DOT1L inhibition led to increased neurogenesis driven by a shift from asymmetric self-renewing to symmetric neurogenic divisions of APs. At the molecular level, we show that DOT1L activity preserved AP identity by promoting transcription of a gene set involved in AP metabolism. On a mechanistic level, DOT1L inhibition increased expression of metabolic genes, including microcephaly-associated Asparagine synthetase (Asns) and overexpression of ASNS in APs resulted in increased neuronal differentiation. Asns expression was predicted to be controlled through EZH2 and we show that DOT1L activity allows PRC2-mediated repression of Asns expression. Importantly, inhibition of ASNS activity rescued increased AP differentiation upon DOT1L inhibition. Our data show that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating AP metabolism, and they provide a mechanistic view on how DOT1L activity might affect neocortical neurogenesis.

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