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Topoisomerase Function in genome and epigenome regulation during Neuronal Differentiation

Final Report Summary - TOPONEURONAL (Topoisomerase function in genome and epigenome regulation during neuronal differentiation)

My original research proposal aimed at addressing the following key questions:
1. Is Top2ß required for terminal differentiation of embryonic stem cells? Does it contribute to the expression of certain genes?
2. Does it regulate the expression of target genes by directly interacting with genes promoters? What are the genomic sites targeted by Top2ß genomewide? Is there any specific epigenetic signature that associates with these regions?
3. Does Top2ß contribute to transcriptional regulation via regulation of higher order chromatin structure?

1. We show that a switch in the expression from Top2a to ß occurs during neuronal differentiation of stem cells in vitro during the transition from a proliferative to a postmitotic state. We derived Top2ß -/- ES cells from the crossings of Top2ß +/- mice. Top2ß -/- ES cells were indistinguishable from wild type cells. Further, Top2ß deficiency does not impair differentiation into neuronal progenitors but results in premature death of neurons. We further assessed genome-wide changes in gene expression for various stages of differentiation for Top2ß KO versus wild type stem cells, neuronal progenitor cells and terminal neuronal cells and discovered that the maximum number of genes were affected at the terminal neuronal stage, associating with the neuronal degeneration phenotype. These results suggested that Top2ß likely has a role in regulating the expression of certain genes that may be pivotal for terminal neuronal differentiation.

2. We then queried whether Top2ß directly interacts with chromatin. We tested a range of anti-Top2ß antibodies and established a chromatin immunoprecipitation (ChIP) protocol that gives the best possible enrichments for its genomic targets. However, although Realtime-PCR results indicated clear enrichment at certain anticipated targets, the enrichments levels were not high enough for Deep-seq experiments. This led us to carry out ChIP-chip assays and we hybridized Top2ß ChIP DNA to NimbleGen HD2.1 arrays containing promoter regions of all known genes including the entire chromosome 19. Genome-wide binding analysis revealed that Top2ß binds preferentially to promoter regions in the genome, and correlates highly with the active chromatin mark H3K4me2. Top2ß bound genes that are down-regulated in Top2ß knockout cells are preferentially associated with the GO term 'neuronal development' while those Top2ß bound and up-regulated in Top2ß kock-out cells associated with a broader GO term 'organismal development'. A significant number of Top2ß targets are also down-regulated upon ICRF-193 mediated inhibition of Top2ß activity, both in in vitro derived neuronal cells and in primary cortical neurons. We could further identify up-regulation of Ngfr P75, a gene directly bound and repressed by Top2ß, as the causal event for degeneration of Top2ß knockout neurons.

3. The above observations supported our hypothesis that Top2ß regulates the expression of target genes by directly modulating their promoters. Our work has further suggested that Top2ß is mostly involved in the later stages of neuronal maturation and is not important for early stages of differentiation and neurogenesis. Studying whether Top2ß-mediated transcriptional control at gene promoters involves modulation of higher order chromatin architecture would be very interesting. However, due to challenging nature of the techniques involved and material source (neurons), this is not yet done. However, we have been able to show that up-regulation of Ngfr P75, a gene bound and repressed by Top2ß, mediates the degeneration of Top2ß knock-out neurons.

Overall, our findings uncover genome-wide binding sites for Top2ß, thereby providing new insights into the regulatory function for Topoisomerases in post-mitotic cells. We put forth a model whereby Top2ß occupies active regulatory regions in the genome where its catalytically activity participates in gene regulation.

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