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.
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: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:Abnormal trophoblast invasion is associated with the most common and most severe complications of human pregnancy. The biology of invasion, as well as the etiology of abnormal invasion remains poorly understood. The aim of this study was to characterize the transcriptome of the HTR-8/SVneo human cytotrophoblast cell line which displays well characterized invasive and non-invasive behaviors, and to correlate the activity of the transcriptome with nuclear matrix attachment and cell phenotype. Interestingly comparison of the transcriptome did not reveal an obvious significant difference between the transcriptomes of invasive and non-invasive HTR cells. In contrast, comparison of the MARs on chromosomes 14-18 revealed an increased number of MARs associated with an invasive phenotype. These attachment areas were more likely to be associated with silent (rather than actively transcribed) genes. DNA extraction with a 2 M NaCl solution followed by restriction digestion with EcoR1 facilitates the separation of matrix bound DNA from non matrix bound DNA. Genomic CGH arrays were used to map the relative differences between attached (scaffold/matrix) and non-attached (loop) portions of the genome in non-invasive (proliferative) and invasive trophoblasts. The matrix association was assessed relative to gene expression measured on Illumina expression beadchips.
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.