Dobson, Jason R.

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. J. Cell. Physiol. 232: 1295–1305, 2017. © 2016 Wiley Periodicals, Inc.

The Brahma (BRM) and Brahma‐related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi‐subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells. J. Cell. Physiol. 9999: 2683–2694, 2015. © 2015 Wiley Periodicals, Inc.