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The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division

Jonathan Pettitt1, Catriona Crombie1, Daniel Schümperli2 and Berndt Müller1,*

1 Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
2 Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland



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  		Fig. 1. C. elegans histone hairpin-binding protein and the histone hairpin RNA sequence element. (A) C. elegans HBP sequence aligned with the RNA-binding domains (RBD) of other HBPs. Both the full-length protein and the RBD (underlined) bind to the C. elegans histone hairpin structure (Michel et al., 2000Go). (B) The conserved sequence element in the 3' untranslated region of histone genes encompassing the hairpin structure. Conserved residues in metazoan histone RNA hairpins are circled. C11 is unique to C. elegans histone RNAs. Nucleotide position numbering is arbitrary.

 


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  		Fig. 2. R06F6.1::gfp is expressed in all somatic cells throughout development. (A) Confocal Z-series projection of an embryo approximately 300 minutes after the first cell division. (B) An epifluorescent image of the expression of R06F6.1::GFP in an L2 larva. The actively dividing nuclei of the lateral hypodermal cells are indicated by arrowheads, and an intestinal nucleus (which undergoes endoreduplication during postembryonic development) is indicated by an arrow. These cells show stronger GFP expression than non-dividing cells. (C) Epifluorescence image showing that nuclei of recently divided cells strongly express R06F6.1::GFP. The cells that form the postdeirid (boxed) and two lateral hypodermal cells (arrows) show stronger expression than neighbouring cells. Scale bars represent 10 µm.

 


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  		Fig. 3. Depleting HBP levels in the early embryo results in defects in chromatin structure. (A) A DIC photomicrograph of a wild-type embryo immediately after the division of the AB and P1 blastomeres, which generates ABa/ABp and EMS/P2, respectively. (B) An epifluorescent image of the same embryo in A showing the H2B::GFP expression associated with the newly divided nuclei. The arrowhead indicates one of the polar bodies that has been drawn between the two AB nuclei upon cytokinesis (the other polar body is visible at the anterior of the embryo). (C) A DIC photomicrograph of an embryo derived from an animal raised on R06F6.1 dsRNA expressing HT115 bacteria at an equivalent developmental stage to the embryo in (A,B). (D) An epifluorescence image of the same embryo as in (C), with H2B::GFP labelling the chromatin. Arrow indicates fluorescent material present between the dividing cells. Note the presence of the polar body (arrowhead), which indicates that this cell has undergone cytokinesis. (E) A DIC photomicrograph of a wild-type 4-cell embryo. Note that all four blastomeres have round nuclei. (F) A DIC photomicrograph of a his-9/10(RNAi) 4-cell embryo showing abnormal nuclear morphology in the two AB daughter cells (white arrowheads). (G,H). Epifluorescence images of wild-type (G) and R06F6.1(RNAi) (H) fixed embryos stained with DAPI. Arrows indicate cells in anaphase showing the poorly condensed chromosomes of R06F6.1(RNAi) embryos compared to wild- type. Anterior is to the left in all panels. Scale bar represents 10 µm.

 


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  		Fig. 4. Late larval lineages are affected by depleting HBP expression during postembryonic development. DIC photomicrographs of postembryonic developmental stages are shown. (A) A vulva and uterus of a wild-type mid-L4 stage larva, showing the so-called `Christmas tree' morphology of the vulva at this developmental stage and obvious uterine cavity (ut). The arrow indicates the lamina that separates the uterine cavity from the vulva. (B) Abnormal vulva and uterus in a mid-L4 stage larvae fed R06F6.1 dsRNA from hatching is shown. Vulval morphology is grossly abnormal and the uterine cavity is completely absent, owing to multiple failures in the execution of uterine development. Note also the presence of germline nuclei proximal to the vulva. (C) Adult wild-type vulva. (D) Evl phenotype of adult fed R06F6.1 dsRNA from hatching. (E) Wild-type male tail, showing the spicules (arrow). Sensory rays are not visible in this focal plane. (F) R06F6.1(RNAi) male tail showing crumpled spicules (arrow). Scale bars represent 10 µm in (A,B,E,F) and 20 µm in (C,D).

 


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  		Fig. 5. HBP is required for histone gene expression. (A) Extracts prepared from wild-type and R06F6.1(RNAi) C. elegans were analysed on a 15% SDS-PAGE. Proteins were detected by western blotting using antibodies against histone H3 (lanes 1, 2) or histone H4 (3, 4), as well as with an anti-tubulin antibody to control for loading. (B,C) Epifluorescence image showing immunostaining with anti-histone H3 antibody of a wild-type (B) and an R06F6.1(RNAi) embryo (C). Scale bars represent 10 µm.

 


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  		Fig. 6. his-9/10(RNAi) causes defects in vulval precursor cell fate. (A) A DIC photomicrograph of the central region of a wild-type adult hermaphrodite. (B) A DIC photomicrograph of a his-9/10(RNAi) adult hermaphrodite displaying a Muv phenotype. Arrowheads indicate the vulva in both animals. The ectopic pseudovulvae are indicated by asterisks. Scale bar represents 50 µm.

 

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