Identification of transcription factor- and gene-interactions of those WSTF- and ISWI-chromatin remodelling complexes that are important in regulating neural stem cell identity

The potential of neural stem cells to differentiate into all neural tissues explains their appeal for therapies against neurological disorders. To optimise the use of neural stem cells for therapeutic purposes, a better understanding is needed of the molecular pathways underlying the different cell-fate decisions that neural stem cells can take. Recent reports suggest that the chromatin structure in embryonic stem cells (ESCs) changes not only at a local (gene) level but also at a global level upon differentiation into neural stem cells (NSCs). It is therefore not surprising that a chromatin remodelling complex such as the ISWI complex can regulate the balance between self-renewal and differentiation in neural stem cells in zebrafish, and affect neural development in Xenopus. The underlying molecular circuitry of this ISWI function is, however, unknown. ISWI-interacting protein WSTF (Williams Syndrome Transcription factor) is part of a heterozygous deletion on human chromosome 7 that causes Williams Syndrome (WS), a developmental and neural retardation syndrome that affects 1 in 20000 people. WSTF was suggested to be involved in some aspects of WS. WSTF localises specifically to neural tissues in developing Xenopus embryos, and a WSTF-ISWI complex is therefore a prime candidate complex for the observed neural phenotype in ISWI mutants. Here I propose to identify (transcription) factors that specifically bind to WSTF, or ISWI-isoform SNF2H, in NSCs, as compared to ESCs. We will use one-step FLAG-affinity purification in combination with mass spectrometry-mediated identification of the co-purifying interaction partners. Selected interaction partners will be verified by co-immunoprecipitation using WSTF- or SNF2H-antibodies. Verified interaction partners and WSTF and SNF2H themselves, will be tested for a role in in vitro ESC differentiation into NSCs, NSC-self renewal, and NSC differentiation into several types of neurons and glial cells, using RNA-interference-mediated depletion of the protein of interest. WSTF, SNF2H and interactors with a phenotype in NSCs will be further investigated by measuring changes in gene expression using micro-arrays in NSCs that are knocked-down for the target protein by RNA interference. Genes that alter their gene expression in the knock-down NSCs will be tested for direct interaction of WSTF/SNF2H and the interactor with the promoter region of the gene, using chromatin-immunoprecipitation. Direct target genes of WSTF or SNF2H isolated in this way will be tested for changes in chromatin structure and replication timing upon action of the WSTF-SNF2H complex. WSTF and SNF2H and their selected interactors will also be knocked-down in Zebrafish, using morpholino oligos, to study their impact on brain development. We will specifically look at the proliferation of NSCs and their differentiation into neural cells, using in situ hybridisation with cell-lineage specific markers on developing knock-down zebrafish embryos. Although the prime purpose of the proposal is to understand the role of WSTF-ISWI in neural stem cell identity, and thereby perhaps learn more about the role of WSTF in Williams Syndrome, it also addresses a number of other issues, such as the role of chromatin in stem cell identity, and the biological function of ISWI complexes. ISWI complexes are abundantly present during early development but their biological relevance is poorly understood, due to the lack of knowledge on the pathways in which they act, and their target genes. From a technical perspective, successful purification and identification of protein complexes from ES cells was reported from only one lab in the world and none in Europe. No protein-complex purifications from neural stem cells were ever reported. Funding of this proposal is therefore also an investment to expand and further optimise technology in which my lab is at the forefront in the world, and which will be essential if we want to understand the interaction-network of protein-factors that give stem cells their unique qualities.