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 histonebinding 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.Eukaryotes package their genomic DNA into a complex nucleoprotein structure referred to as chromatin. The fundamental repetitive element of chromatin is the nucleosome, which is composed of 146 bp of DNA wrapped around an octamer of histone proteins: two dimers of histone H2A and H2B and a tetramer of histones H3 and H4 (56). This chromatinized template DNA is the substrate for in vivo reactions such as transcription, replication, recombination, and repair. Indeed, proper chromatin assembly or modification is necessary for the accurate execution and regulation of these processes.The cell uses a number of mechanisms to build nucleosomes. Nucleosomes are assembled in a DNA replication-coupled (RC) manner following the replication fork during S phase and are assembled onto DNA during gap-repair in response to DNA damage (28,35). In both cases, nascent DNA is packaged into chromatin. Alternatively, it has been demonstrated that nucleosomes can be assembled or reassembled independently of DNA replication. Replication-independent (RI) assembly is not limited to the S phase but occurs continually throughout the cell cycle (2, 4), perhaps functioning as a backup to RC assembly (44). RI assembly can introduce specific histone variants, such as the H3 variant Cid, at centrosomes (1) or histone H3.3 in transcriptionally active regions of the genome (2). By extension it has been proposed that RI assembly may replace histones that have been irreversibly modified by methylation (23); otherwise, the only way to change the histone methylation signal would be by gradual dilution of methylated with unmethylated histones in the course of cell proliferation. RI assembly also occurs in nondividing cells. For example, in nerve cells of higher eukaryotes infected with herpes simplex virus, RI assembly quickly packa...

The structure of nucleosome assembly protein 1

“…Members of the NAP family have been shown to bind to a variety of proteins involved in transcription, cell cycle regulation, protein shuttling, chromatin assembly, histone variant exchange, and chromatin remodeling function (12,16,34,43,44). Drosophila NAP-1 coprecipitates with core histones H2A-H2B from embryos (5).…”

The Chd family of chromatin remodelers

“…Biochemical analyses revealed that ScChd1 has an ATPase activity that affects DNA-histone interactions within the nucleosome in a manner that is distinct from the yeast SWI/SNF complex [54]. Additionally, a partial loss of chromatin assembly activity in vitro from crude DEAE fractions derived from deletion strains of ScChd1 was observed [55]. Recently, it was shown that ScChd1 preferentially relocated nucleosomes closer to the center of DNA fragments [56].…”

Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II

“…It is unclear whether the physiological role of Spt6 is related to its biochemical activities of nucleosome assembly (7) and/or transcriptional elongation per se (14). Lastly, Chd1 associates with elongation factors and coding regions of actively transcribed genes (49), and it is part of chromatin-modifying and nucleosome assembly activities in vitro (46,55).…”