CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo

Abstract

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.

Fig. 1

Characterization of Chd1 mutant alleles. (A) Genomic structure of the Chd1 locus. Df(2L)JS17 and Df(2L)Exel7014 uncover Chd1. Open boxes, predicted genes. Closed box, Chd1. Arrows, chromosome deficiencies. Dashed lines, deficiency breakpoints. Triangle, P{EPgy2}EY07345 insertion that was used for excisions. (B) The Chd1[1] and Chd1[2] excisions delete 296 and 958 amino acids, respectively, from the C-terminus of CHD1. Chd1[3] has a nonsense mutation resulting in a stop at Q1394. The distal breakpoint of Df(2L)Exel7014 is located immediately downstream of the Chd1 3′-UTR. Open boxes, predicted genes. Closed box, Chd1 coding sequence. Gray box, Chd1 ATPase domain. (C) Western blot of heterozygous mutant embryos. Truncated CHD1 polypeptides are not detected in heterozygous Chd1[1] or Chd1[2] embryos. Heterozygous Chd1[3] embryos express a truncated (residues 1–1394) CHD1 polypeptide. Arrowhead, wild-type CHD1 (250 kDa). Arrow, NAP-1 (loading control). CyO, second-chromosome balancer.

Fig. 2

Embryos from homozygous Chd1 mutant females are haploid. (A) Propidium iodide (PI) staining reveals the haploid chromosome content in Chd1 null embryos (right). Cycle 10 embryos are shown. (B) Propagation of only the maternal genome is detected by PCR in embryos from Chd1 females that have been mated with males carrying a GFP transgene. Primers for GFP recognize the paternal DNA, primers for Asf1 amplify sequences from both male and female genomes. (C) The absence of maternal CHD1 results in the inability of one pronucleus (arrows) to enter the first mitosis. The other pronucleus (arrowheads) continues with divisions (left, prophase – metaphase; right, post-anaphase). Labeling above the panels refers to genotypes of mothers. Scale bars, 10 μm.

Fig. 3

CHD1 is required for incorporation of histones into decondensing sperm chromatin. (A) Histone H3 co-localizes with DNA in both parental pronuclei of wild-type embryos (left, interphase/prophase; right, metaphase). (B) In Chd1 mutant embryos, the male pronucleus fails to undergo mitosis and accumulates abnormally little H3. (C) H3.3-FLAG is incorporated into chromosomes of the male pronucleus in wild-type embryos. Panels show the first metaphase. (D) In Chd1 mutant eggs, H3.3-FLAG accumulates in the periphery of the male pronucleus. The female pronucleus proceeds with mitosis (left, prophase; right, anaphase). Arrows, male pronuclei (m). Arrowheads, female pronuclei (f). Labeling above the panels refers to genotype of mothers. Red, PI; green, H3 (A and B) or H3.3-FLAG (C and D). Scale bars, 10 μm.

Fig. 4

Deposition of H3.3 into chromatin during syncytial blastoderm is compromised in Chd1 mutants. (A) H3.3-FLAG overlaps with DNA in syncytial nuclei of a cycle 14 wild-type embryo. (B) H3.3-FLAG co-localizes poorly with DNA in Chd1 mutant embryos. Labeling above the panels refers to genotypes of mothers. Red, PI; green, H3.3-FLAG. Scale bars, 10 μm.