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, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we mapped 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate from HeLa cells across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites recovered mapped predominately near expressed genes and localized near transcription start sites and the ends of genes.
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. 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 AoAF cells.
Project description:Nuclear chromosomes transcribe far more RNA than required to code for protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin and most nuclear RNA from the nucleus, but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome “scaffold.” RNA-seq analysis reveals most RNA in the nuclear scaffold is repeat-rich, non-coding, and predominantly derived from introns of nascent transcripts. This repeat-rich (C0T-1) RNA inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei, demonstrating RNA physically antagonizes a propensity for chromatin to condense. C0T-1 hnRNA co-distributes on euchromatin with several known scaffold proteins including scaffold attachment factor A (SAF-A). We further show that RNA is required for SAF-A to interact with chromatin and to form structurally embedded scaffold-attachment regions (SARs) in the nuclear genome. Collectively, results indicate nascent transcripts serve a dynamic structural role in the open architecture of active chromosome territories
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: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.