Project description:DNA topoisomerase II (topo II) catalyzes a strand passage reaction in that one duplex is passed through a transient brake in another. Completion of late stages of neuronal development depends on the presence of active isoform (topo IIbeta). We identified topo IIbeta action sites on 7 selected genomic regions (about 79 Mb in total), each containing at least one gene that is controlled by topo IIbeta in neuronal differentiation. We used a novel method, etoposide-mediated topoisomerase immunoprecipitation (eTIP), followed by identification of precipitated DNA fragments on genomic tiling arrays. These DNA fragments were first fractionated by concentrated salt prior to the array analysis. The 0.5 M NaCl-released fraction was analyzed as P2 and the salt-resistant fraction wad analyzed as P1.
Project description:DNA topoisomerase II (topo II) catalyzes a strand passage reaction in that one duplex is passed through a transient brake in another. Completion of late stages of neuronal development depends on the presence of active isoform (topo IIbeta). We identified topo IIbeta action sites on 7 selected genomic regions (about 79 Mb in total), each containing at least one gene that is controlled by topo IIbeta in neuronal differentiation. We used a novel method, etoposide-mediated topoisomerase immunoprecipitation (eTIP), followed by identification of precipitated DNA fragments on genomic tiling arrays. These DNA fragments were first fractionated by concentrated salt prior to the array analysis. The 0.5 M NaCl-released fraction was analyzed as P2 and the salt-resistant fraction wad analyzed as P1. The genomic DNA fractions from the immunoprecipitate (P1 or P2) were co-hybridized with a reference DNA isolated from the input of immunoprecipitation.
Project description:Transcriptome analysis of mRNA samples purified from developing cerebellar granule cells and ES cell-derived granule cells using translating ribosome affinity purification (TRAP) method. Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are up-regulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of up-regulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNA interference in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry. We analyzed gene expression of cerebellar granule cells and ES cell-derived granule cells using the Affymetrix mouse gene 1.0 ST platform. Array data was processed by metagene analysis which was developed by the Broad Institute.
Project description:Transcriptome analysis of mRNA samples purified from developing cerebellar granule cells and ES cell-derived granule cells using translating ribosome affinity purification (TRAP) method. Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are up-regulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of up-regulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNA interference in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry.