Chromatin priming and Hunchback recruitment integrate spatial and temporal cues in Drosophila neuroblasts

Neural stem cells generate diverse cell types by integrating spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. In Drosophila neuroblasts (NBs), spatial patterning sets lineage identity, while a temporal transcription factor (TTF) cascade sets birth order. Two proposed mechanisms could integrate these inputs. In direct regulation, spatial transcription factors (STFs) and TTFs co-occupy and activate TS enhancers within NBs. In epigenetic regulation, STFs first prime NB-specific chromatin, creating sites of integration (SoIs) that later recruit TTFs. We test this in two identified NBs -- NB5-6 and NB7-4 -- and their candidate STFs, Gooseberry (Gsb) and Engrailed (En), together with the first TTF, Hunchback (Hb). In NB5-6, Gsb is expressed transiently, suggesting a chromatin-based memory of its activity. In NB7-4, En expression persists throughout development so integration could either be epigenetic or direct. We used chromatin engagement by the STFs as the discriminator between these models. If integration is epigenetic, the STF must engage less-accessible chromatin to establish NB-specific SoIs; if regulation is direct, the STF need not. We find that En binds only to pre-accessible loci in NB7-4 and En+Hb co-binding marks the most accessible enhancers. This suggests that NB7-4 likely relies on an unknown priming factor to establish SoIs, with direct En-Hb co-binding mediating enhancer activation. In NB5-6, Gsb binds both open and less-accessible chromatin and Gsb+Hb co-binding marks the most accessible enhancers. When ectopically expressed, Gsb remodels chromatin globally in the non-cognate NB7-4, and at endogenous NB7-4 SoIs, it specifically reduces accessibility as well as Hb binding. This suggests that in NB5-6 Gsb likely acts together with other NB5-6-specific factors to recognize less-accessible chromatin and to promote Hb recruitment while restricting Hb occupancy to appropriate enhancers. Together these findings support a unified two-step model: NB-specific combinations of TFs -- each NBs "STF code" -- first prime chromatin and then recruit and restrict Hb to ensure lineage-specific enhancer activation.