Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in HeLa cells. LIS or 2 M NaCl solutions followed by restriction digestion with EcoR1 facilitates the separation from scaffold/matrix bound DNA from non bound DNA. Genomic CGH arrays were used to map the relative differences between attached (scaffold/matrix) and non-attached (loop) portions of HeLa DNA. The expression profile of the HeLa cells used for aCGH analysis was also determined.
Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in AoAF cells. LIS (lithium 3,5-diiodosalicylate) or 2 M NaCl solutions followed by restriction digestion with EcoR1 facilitates the separation from scaffold/matrix bound DNA from non bound DNA. Genomic CGH arrays were used to map the relative differences between attached (scaffold/matrix) and non-attached (loop) portions of AoAF DNA. The expression profile of the AoAF cells used for aCGH analysis was determined.
Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in HeLa cells.
Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in AoAF cells.
Project description:Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.