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Science 24 August 2007:
Vol. 317. no. 5841, pp. 1087 - 1090
DOI: 10.1126/science.1145339
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Reports

CHD1 Motor Protein Is Required for Deposition of Histone Variant H3.3 into Chromatin in Vivo

Alexander Y. Konev1, Martin Tribus2, Sung Yeon Park3, Valerie Podhraski2, Chin Yan Lim4*, Alexander V. Emelyanov1, Elena Vershilova1, Vincenzo Pirrotta3, James T. Kadonaga4, Alexandra Lusser2{dagger} and Dmitry V. Fyodorov1{dagger}

1 Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
2 Division of Molecular Biology, Biocenter, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria.
3 Department of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA.
4 Section of Molecular Biology, University of California at San Diego, La Jolla,CA 92093,USA.


Figure 1 Fig. 1. Characterization of Chd1 mutant alleles. (A) Genomic structure of the Chd1 locus. Df(2L)JS17 and Df(2L)Exel7014 uncover Chd1. White boxes, predicted genes; black box, Chd1; black 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 glutamine 1394. The distal breakpoint of Df(2L)Exel7014 is located immediately downstream of the Chd1 3' untranslated region. White boxes, predicted genes; black 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 to 1394) CHD1 polypeptide. Arrowhead, wild-type CHD1 (250 kD); arrow, left, NAP-1 (loading control); CyO, second-chromosome balancer. [View Larger Version of this Image (29K GIF file)]
 

Figure 2 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 to metaphase; right, post anaphase). Labeling above the panels refers to genotypes of mothers. Scale bars, 10 µm. [View Larger Version of this Image (31K GIF file)]
 

Figure 3 Fig. 3. CHD1 is required for incorporation of histones into decondensing sperm chromatin. (A) H3 colocalizes with DNA in both parental pronuclei of wild-type embryos (left, interphase or 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). (B, C, and D) 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. [View Larger Version of this Image (33K GIF file)]
 

Figure 4 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 colocalizes 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. [View Larger Version of this Image (112K GIF file)]
 

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