Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. In the past year we have made significant progress towards understanding the mechanism of ATP-dependent chromatin remodeling by Drosophila NURF (Nucleosome Remodeling Factor). Previously, we demonstrated that NURF remodels chromatin by inducing nucleosome """"""""sliding,"""""""" the relative movement of a histone octamer without irretrievable displacement from DNA. We have now successfully reconstituted the four-subunit NURF from recombinant proteins, thus allowing analysis of subunit requirements and a mutational study of protein domains. We have found that the two largest NURF components, NURF301 and the ISWI ATPase, are sufficient to reconstitute efficient and accurate nucleosome sliding activity. ISWI is the engine of nucleosome sliding, while NURF301 enhances efficiency and positional specificity of nucleosome movement. To achieve this, NURF301 makes several distinct contacts with nucleosomes. We have defined a HMGA (HMGI/Y)-like DNA-bending domain in NURF301 and demonstrated its functional importance to the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, thus providing a basis for targeted recruitment of the NURF complex to specific genes. We are in position to elucidate the remaining functional domains of NURF301, and to investigate how coupling of NURF301 to the ISWI ATPase leads to nucleosome movements in chromatin. We have isolated mutations for the Drosophila NURF301 gene, and have studied its phenotypes. We are also making excellent progress on studies of a much larger, 12-component chromatin remodeling complex (the INO80 complex). We have completed identification of all 12 subunits by mass spectrometry of tryptic peptides, and are conducting genetic and biochemical experiments to elucidate the mechanism and physiological functions of this new chromatin remodeling complex. Our studies continue to provide new insights to the important link between chromatin and transcription.
