The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo.H istone-DNA interactions constantly change during various processes of DNA metabolism. Recent studies have highlighted the importance of histone variants, such as H3.3, CENP-A (centromere protein A), or H2A.Z, in chromatin dynamics (1, 2). Incorporation of replacement histones into chromatin occurs throughout the cell cycle, whereas nucleosomes containing canonical histones are assembled exclusively during DNA replication. A thorough understanding of the replication-independent mechanisms of chromatin assembly, however, is lacking.In vitro, chromatin assembly requires the action of histone chaperones and adenosine triphosphate (ATP)-utilizing factors (3). Histone chaperones may specialize for certain histone variants. For example, H3.3 associates with a complex containing HIRA, whereas canonical H3 is in a complex with CAF-1 (chromatin assembly factor 1) (4). The molecular motors known to assemble nucleosomes are ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (nucleosome-remodeling and spacing factor), which contain the Snf2 family member ISWI as the catalytic subunit (5-7), and CHD1, which belongs to the CHD subfamily of Snf2-like adenosine triphosphatases (ATPases) (8). These factors have not been shown to mediate deposition of histones in vivo. We previously demonstrated that CHD1, together with the chaperone NAP-1, assembles nucleosome arrays from DNA and histones in vitro (9). Here, we investigated the role of CHD1 in chromatin assembly in vivo in Drosophila.We generated Chd1 alleles by P elementmediated mutagenesis (Fig. 1A) (10). Two exci- 2] with Df(2L)Exel7014 affect both copies of the Chd1 gene only (Fig. 1B). We also identified a single point mutation that results in premature translation termination of Chd1 (Q1394*) in a previously described lethal allele, l(2)23Cd[A7-4] (11). Hence, l(2)23Cd[A7-4] was renamed Chd1 [3].sions, Df(2L)Chd1[1] and Df(2L)Chd1[2], deleted fragments of the Chd1 gene and fragments of unrelated adjacent genes. Heterozygous combinations, however, of Chd1[1] or Chd1[Analysis of Western blots of embryos from heterozyg...

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked ␣-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.Eukaryotic nuclear DNA is compacted into a nucleoprotein complex referred to as chromatin (1). The basic repeating unit of chromatin, the nucleosome, consists of 146 bp of DNA wrapped in 1.7 left-handed superhelical turns around the core histone octamer (2). Different organization levels of chromatin range from 10-and 30-nm filaments in dispersed interphase chromatin to higher order structures in specialized chromosome domains such as telomeres and centromeres to the highly condensed mitotic chromatin fiber. Chromatin is a vastly dynamic entity that varies in structure throughout development and cell cycle progression. Importantly, chromatin is the natural substrate for essential nuclear reactions such as DNA replication recombination, repair, and transcription. The packaging of DNA into various forms of chromatin provides the cell with the means to compact and to store its genetic material and creates an additional level of regulation of the nuclear DNA metabolism.Unlike transcriptionally active, gene-rich, early replicating euchromatin in metazoan chromosome arms, heterochromatin is usually associated with the repression of genetic activity and other enzymatic processes in the nucleus and shelters specialized regions of chromosomes such as centromeres and telomeres (3, 4). Heterochromatin protein HP1a is the major component and diagnostic marker of heterochromatin. In Drosophila polytene chromosomes, HP1a is normally highly concentrated in and near the chromocenter and also associated with telomeres, the fourth chromosome, and a few euchromatic loci (5). Position effect variegation (PEV) 2 is observed when expression of a gene is repressed in a stochastic manner by alteration of its native genomic location, usually by positioning within or near large ...