The human SWI/SNF (hSWI/SNF) ATP-dependent chromatin remodeling complex is a tumor suppressor and essential transcriptional coregulator. SWI/SNF complexes have been shown to alter nucleosome positions, and this activity is likely to be important for their functions. However, previous studies have largely been unable to determine the extent to which DNA sequence might control nucleosome repositioning by SWI/SNF complexes. Here, we employ a minicircle remodeling approach to provide the first evidence that hSWI/SNF moves nucleosomes in a sequence dependent manner, away from nucleosome positioning sequences favored during nucleosome assembly. This repositioning is unaffected by the presence of DNA nicks, and can occur on closed-circular DNAs in the absence of topoisomerases. We observed directed nucleosome movement on minicircles derived from the human SWI/SNF-regulated c-myc promoter, which may contribute to the previously observed "disruption" of two promoter nucleosomes during c-myc activation in vivo. Our results suggest a model wherein hSWI/SNF-directed nucleosome movement away from default positioning sequences results in sequence-specific regulatory effects.
During normal hemostasis, the coagulation protease factor (F)XIa activates FIX. Hereditary deficiency of the FXIa precursor, FXI, is usually associated with reduced FXI protein in plasma, and circulating dysfunctional FXI variants are rare. We identified a patient with < 1% normal plasma FXI activity and normal levels of FXI antigen, who is homozygous for a FXI Gly555 to Glu substitution. Gly555 is two amino acids N-terminal to the protease active site serine residue, and is highly conserved among serine proteases. Recombinant FXI-Glu555 is activated normally by FXIIa and thrombin, and FXIa-Glu555 binds activated factor IX similarly to wild type FXIa (FXIa(WT)). When compared with FXIa(WT), FXIa-Glu555 activates factor IX at a greatly reduced rate ( approximately 400-fold), and is resistant to inhibition by antithrombin. Interestingly, FXIa(WT) and FXIa-Glu555 cleave the small tripeptide substrate S-2366 with comparable k(cat)s. Modeling indicates that the side chain of Glu555 significantly alters the electrostatic charge around the active site, and would sterically interfere with the interaction between the FXIa S2' site and the P2' residues on factor IX and antithrombin. FXI-Glu555 is the first reported example of a naturally occurring FXI variant with a significant defect in FIX activation.