Formation of the CNS during embryogenesis requires the de novo initiation of neural gene expression in embryonic ectoderm. E.g., the Hoxb1a and Hoxb1b transcription factors (TFs) are required for zebrafish hindbrain development, but it is unclear how they activate gene expression. In particular, embryonic chromatin is compacted, making regulatory elements inaccessible to most TFs. We find that Prep and Pbx (two TFs identified as cofactors to Hox proteins) occupy genetic loci prior to zygotic genome activation – many hours before Hox proteins are active and well before hindbrain development is initiated. Also, Prep:Pbx occupied sites co-localize with binding sites for the NFY TF. We hypothesize that maternally deposited Prep:Pbx cooperates with NFY to open chromatin at hindbrain genes. This process exposes binding sites for hindbrain-specific TFs (e.g. Hox proteins) to drive gene expression in the hindbrain. There are three aims of our proposed work: First we will disrupt Prep:Pbx and NFY function to determine if they modulate the chromatin state. Second, we will determine if Prep:Pbx-mediated chromatin changes promote binding by Hoxb1b to create hindbrain- specific enhancers. Lastly, we will determine how such enhancers drive hindbrain-specific gene expression. This work is relevant for several reasons. First, it is important to understand hindbrain development per se (as defects give rise to ataxias etc.). Second, our results will be applicable to understanding hox function in general, which is important since other hox genes are involved in hindbrain development (and many other cell fate decisions). Additionally, prep and pbx are proto-oncogenes and it is likely that their ability to drive de novo gene expression will explain some of their oncogenic potential. Lastly, our experiments will show how genes can be activated in inaccessible chromatin. This will be applicable to targeted differentiation/reprogramming of stem or precursor cells – hence our work will also inform efforts to generate specific cell types for clinical applications.