The vast majority of the neurons comprising the brain are derived from Neural Stem Cells (NSCs). Like other stem cells, NSCs must balance their defining, yet seemingly opposing, features of self-renewal and the ability to terminally differentiate into neurons or glia. How this exquisite balance is achieved during neurogenesis remains unclear. The long-term goal of the proposed project is to understand the transcriptional mechanisms that govern NSC identity in vivo. This requires identifying NSC determinants and gaining mechanistic insight into how they function individually and how they are integrated within a transcriptional network that maintains its robustness over multiple cell divisions. While NSC identity genes that encode epigenetic regulators or classical transcription factors have garnered much attention in recent years, our unpublished work and that of others increasingly points to a highly selective role of general transcription factors in stem cell identity. In particlar, we have identified a unique subset of TATA-box binding protein (TBP)-associated factors (TAFs) and a TBP paralog, TRF2, but not TBP itself, as novel NSC identity genes. Because TAFs were first identified by virtue of their association with TBP, they are often regarded as general transcription factors and assumed to be required for the bulk of transcription. In contrast, our preliminary studies have shown that a unique subset of TAFs (NSC-TAFs) and TRF2 are selectively required for NSC properties in vivo. Knockdown of NSC-TAFs or TRF2 leads to premature differentiation, cell cycle progression defects, and ectopic accumulation of the pro- differentiation factor Prospero/Prox1 in the NSC nucleus. In addition, while NSCs mutant for TAF7 proliferate poorly and prematurely differentiate, prospero, Taf7 double mutant NSCs generate large NSC clones that are mostly composed of cells expressing the NSC marker Asense. This indicates that removing prospero restores stem cell properties to TAF7 mutant NSCs and that Prospero is epistatic to TAF7. Our central hypothesis is that NSC-TAFs and TRF2 form a novel complex that selectively associates with, and is required for, the expression of a novel subset of NSC-expressed genes with shared core promoter architecture. To test this, we propose to leverage recent, sophisticated transcript and chromatin profiling techniques available in Drosophila to probe the molecular basis of control of NSC identity by NSC-TAFs and TRF2. Specifically, in Aim 1 we will purify NSCs and perform RNA-seq to identify transcripts that require both NSC-TAFs and TRF2 for NSC expression.
In Aim 2 we will profile the genomic binding sites of NSC-TAFs and TRF2 in NSCs in vivo and attempt to identify a NSC-TAF-TRF2 complex, using both co-immunoprecipitation and proximity ligation assays. Overall, our work will allow us to gain a foothold on the molecular mechanisms that control NSC identity through NSC-TAFs and TRF2. Importantly, TAF mutations have been linked to diverse human neurological disorders and we expect our proposed research will shed light on disease pathogenesis, inform modeling of TAF-linked neurological disorders, and guide NSC-based therapies.
Neural stem cells (NSCs) are the building blocks of the brain in many animals, including humans. Defects in genes that control NSCs have been linked to neurological disorders and this study proposes to use novel genomic tools to decipher how genes work together to maintain NSCs in an undifferentiated state. Such knowledge is crucial for rational design of NSC-based therapies for human disease.