THE DYNAMIC INTERPLAY BETWEEN HOMEODOMAIN TRANSCRIPTION FACTORS AND CHROMATIN ENVIRONMENT REGULATES PRONEURAL FACTOR OUTCOMES

Generation of neurons of vast neurotransmitter, morphological and hodological subtype diversity involves early spatial and temporal patterning of the neuronal precursors by morphogenic gradients and combinatorial expression of homeotic transcription factors. Proneural basic-helix-loop-helix (bHLH) transcription factors play a key role in this process. However, it remains unknown how bHLH factors such as NGN2 and Ascl1, which are primarily proneural, contribute to subtype specification in a cell context-dependent manner. Here, we investigate the context-dependent molecular function of a pro-neural bHLH transcription factor NGN2; a factor which we previously showed to efficiently induce the neuronal fate in human embryonic stem (hES) cells. We tested its molecular function and lineage-inducing capacity in context of different chromatin environments and in combination with other transcription factors with spatially restricted expression patterns and evaluated NGN2 genomic localization, chromatin dynamics and transcriptional consequences using multi-omics methods. This work revealed that the combination of NGN2 and EMX1, a forebrain-restricted homeobox transcription factor, yields a highly homogeneous glutamatergic neuronal forebrain subpopulation without partial cholinergic and monoaminergic gene programs seen in NGN2 only cells. In contrast, modifying the chromatin environment that NGN2 is exposed to, strongly regulates the regional identity of resulting neurons that persists throughout reprogramming but does not influence neurotransmitter subtype specification. This shows how nature co-opts combinatorial strategies of using different chromatin landscapes and/or homeodomain transcription factors with spatially restricted expression to generate a vast variety of neuronal subtypes with a few transcription factors. Our work thus provides improved strategies to generate a plethora of defined and subtype specific neuronal cell populations from pluripotent stem cells for therapeutic or disease modeling purposes.