Human SWI/SNF directs sequence-specific chromatin changes on promoter polynucleosomes.

Studies in humans and other species have revealed that a surprisingly large fraction of nucleosomes adopt specific positions on promoters, and that these positions appear to be determined by nucleosome positioning DNA sequences (NPSs). Recent studies by our lab, using minicircles containing only one nucleosome, indicated that the human SWI/SNF complex (hSWI/SNF) prefers to relocate nucleosomes away from NPSs. We now make use of novel mapping techniques to examine the hSWI/SNF sequence preference for nucleosome movement in the context of polynucleosomal chromatin, where adjacent nucleosomes can limit movement and where hSWI/SNF forms altered dinucleosomal structures. Using two NPS templates (5S rDNA and 601) and two hSWI/SNF target promoter templates (c-myc and UGT1A1), we observed hSWI/SNF-driven depletion of normal mononucleosomes from almost all positions that were strongly favored by assembly. In some cases, these mononucleosomes were moved to hSWI/SNF-preferred sequences. In the majority of other cases, one repositioned mononucleosome appeared to combine with an unmoved mononucleosome forming a specifically localized altered or normal dinucleosome. These effects result in dramatic, template-specific changes in nucleosomal distribution. Taken together, these studies indicate hSWI/SNF is likely to activate or repress transcription of its target genes by generating promoter sequence-specific changes in chromatin configuration.

In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth.

Human aortic endothelial (HAEC) and human coronary artery smooth muscle cell (HCASMC) responses on electrospun silk fibroin scaffolds were studied to evaluate potential for vascular tissue engineering. Cell proliferation studies supported the utility of this biomaterial matrix by both HAECs and HCASMCs. Alignment and elongation of HCASMCs on random non-woven nanofibrous silk scaffolds was observed within 5 days after seeding based on SEM and confocal microscopy. Short cord-like structures formed from HAECs on the scaffolds by day 4, and a complex interconnecting network of capillary tubes with identifiable lumens was demonstrated by day 7. The preservation of cell phenotype on the silk fibroin scaffolds was confirmed by the presence of cell-specific markers, including CD146, VE-cadherin, PECAM-1 and vWF for HAECs, and SM-MHC2 and SM-actin for HCASMCs at both protein and transcription levels using immunocytochemistry and real-time RT-PCR, respectively. Formation of ECM was also demonstrated for the HCASMCs, based on the quantification of collagen type I expression at protein and transcription levels. The results indicate a favorable interaction between vascular cells and electrospun silk fibroin scaffolds. When these results are factored into the useful mechanical properties and slow degradability of this protein biomaterial matrix, potential utility in tissue-engineered blood vessels can be envisioned.