Loss of the basic helix-loop-helix transcription factor Bhlhe41 induces cell death and impairs neurite outgrowth in Neuro2a cells.

The basic helix-loop-helix (bHLH) superfamily of transcription factors have been implicated in a wide range of cellular functions such as proliferation, differentiation, tumorigenesis, and circadian rhythms. In a previous siRNA-based screen, bHLH family member e41 (BHLHE41) had been identified as a putative regulator of neuronal differentiation; however, its function remains largely elusive. To this end, using the CRISPR-Cas9 system, we established an isogenic Neuro2a (N2a) cell line with biallelic targeting of Bhlhe41 gene (Bhlhe41-/-). In undifferentiated N2a cells, complete knockout of Bhlhe41 resulted in marked proliferation inhibition, together with accumulation of apoptotic cells. Furthermore, retinoic acid (RA)-induced neurite outgrowth and expression of neuronal markers are significantly weakened in Bhlhe41-/- cells. We also showed that the activity of ERK1/2 signaling, a key regulator of neuronal differentiation, is likewise impaired in knockout cells. Together, these results suggest that Bhlhe41 plays critical roles in regulating cell death and neurite outgrowth in N2a cells.