Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and α-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.     

Structural analysis of the yeast SWI/SNF chromatin remodeling complex

Elucidating the mechanism of ATP-dependent chromatin remodeling is one of the largest challenges in the field of gene regulation. One of the missing pieces in understanding this process is detailed structural information on the enzymes that catalyze the remodeling reactions. Here we use a combination of subunit radio-iodination and scanning transmission electron microscopy to determine the subunit stoichiometry and native molecular weight of the yeast SWI/SNF complex. We also report a three-dimensional reconstruction of yeast SWI/SNF derived from electron micrographs.     

SWI/SNF chromatin remodeling requires changes in DNA topology

ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.     

Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits

Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.     

Human SWI/SNF nucleosome remodeling activity is partially inhibited by linker histone H1

The physical structure and the compact nature of the eukaryotic genome present a functional barrier for any cellular process that requires access to the DNA. The linker histone H1 is intrinsically involved in both the determination of and the stability of higher order chromatin structure. Because histone H1 plays a pivotal role in the structure of chromatin, we investigated the effect of histone H1 on the nucleosome remodeling activity of human SWI/SNF, an ATP-dependent chromatin remodeling complex. The results from both DNase I digestion and restriction endonuclease accessibility assays indicate that the presence of H1 partially inhibits the nucleosome remodeling activity of hSWI/SNF. Neither H1 bound to the nucleosome nor free H1 affected the ATPase activity of hSWI/SNF, suggesting that the observed inhibition of hSWI/SNF nucleosome remodeling activity depends on the structure formed by the addition of H1 to nucleosomes.