Chromatin factor regulation of neuroblast progenitor genome dynamics

PROJECT SUMMARY One of the fundamental questions in development is how cellular diversity arises from a single genome template. The metazoan genome non-randomly organized within nuclei, and this organization is thought to underlie cell type specific gene expression. In brain development, how genome organization relates to cell identity may be a particularly challenging question, as neural progenitors sequentially give rise to distinct neural cell types, and the incredible cellular diversity arises from a limited progenitor pool. Neural progenitors transit through distinct states of competence, or potential, which restricts the period they can generate each cell type; this ordered production of neurons gives rise to the organized tissue structure underlying brain function. How neural progenitor competence is regulated and how it changes over time is not understood. At the interface of neurodevelopment and genome architecture, we are studying mechanisms of competence regulation in vivo by asking how the three dimensional organization of the neural progenitor genome contributes to gene expression in the neuron. While progress in high throughput sequencing has revealed the basic principles of genome folding at multiple scales, what level of organization ultimately relates to gene expression and cell function remain mostly unknown. This is largely because genome organization is highly context specific, and cell type and stage specific genomic data is challenging to obtain and to validate in vivo. Further, there is a dearth of knowledge of genome architecture regulators and the mechanisms of their functions. We have established Drosophila embryo neuroblasts (NBs, fly neural progenitor) as model to study how genome organization relates to NB competence in vivo. We found that the hunchback (hb) gene, a master regulator gene for early-born neurons, relocates to the NB nuclear periphery at mid-embryogenesis. This relocation event within the NB progenitor heritably silences the hb gene, rendering it refractory to activation in the descendent neuron. Thus, changes in hb gene positioning within the NB terminates competence to specify early-born molecular identity of the neuron. We discovered a cis-acting gene mobility element within the hb intron that is both necessary and sufficient for hb gene relocation to the NB nuclear lamina, a first clue in understanding how genes mobilize relative to the nuclear periphery. We have also identified multiple trans-acting factors that regulate hb gene relocation in vivo, laying the groundwork to test how distinct nuclear architecture regulators orchestrate developmentally-timed genome reorganization. Here we will purify NBs from different developmental stages to profile genome-wide chromatin interactions in vivo and examine the role of NB nuclear factors in genome architecture reorganization. We will also test how the phase-separation properties of one such NB factor contributes to the timing of genome reorganization. Together, our proposed studies will generate new mechanistic insights underlying NB genome architecture dynamics, the role that trans-acting factors play to coordinate the timing of genome reorganization, and ultimately how these changes in genome structures impact specification of neural identity.