Replication-Independent Assembly of Nucleosome Arrays in a Novel Yeast Chromatin Reconstitution System Involves Antisilencing Factor Asf1p and Chromodomain Protein Chd1p

Abstract

Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor. Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution. In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease. We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo.

ACKNOWLEDGMENTS

Some reagents used in this study were prepared by K.M.R. in the lab of Jim Kadonaga, whom we thank for advice, support, and provision of materials. We also thank Paul Laybourn for the β-endoglucanase gene and, along with John Pilon, for advice on yeast histone purification and the generous gift of recombinant yeast histones. In addition, we thank Dmitry Fyodorov, Mark Levenstein, Mike Pazin, and Jessica Tyler for useful discussions and David Stuart for the generous gift of yeast strain DSY904 and for critical reading of the manuscript.

This work was funded by an operating grant to M.C.S. from the Canadian Institutes for Health Research. M.C.S. is a Scientist of the Alberta Heritage Foundation for Medical Research.