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Fig. 1 | Epigenetics & Chromatin

Fig. 1

From: Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-AHCP−3 Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes

Fig. 1

Newly formed ACs in C. elegans are enriched with histone H3K9 and H4 acetylations, which decline gradually in several embryonic cell divisions. a A schematic diagram of the experimental setup. Plasmid DNA with 64 copies of LacO repeats (p64xLacO) and a dominant roller mutant allele which express in larva and adults {pRF4[rol-6(su1006)]} was mixed and co-injected into strains expressing either GFP::LacI (under pie-1 promoter) or GFP::LacI::HDA-1 (under hda-1 promoter). Injected DNA concatemerized to form artificial chromosomes (ACs) in 1-cell embryos produced by injected worms. b On the left, a representative image of immunofluorescence of LacI (green) and DAPI (blue) is shown separately and merged in 1 or 2-cell stage embryos in GFP::LacI-tethering strain without or with DNA injection. Scale bar represents 10 μm. Examples of endogenous chromosomes in the nucleus are highlighted in yellow rectangles. LacI staining is nuclear in the uninjected case and forms foci and with weaker nuclear staining in the injected case. A representative AC is highlighted in a red rectangle in the injected case. On the right, a representative merged image of immunofluorescence of LacI (green) and DAPI (blue) in a 9 to 16-cell stage embryo in GFP::LacI-tethering strain after DNA injection is shown. Scale bar represents 10 μm. A cell with an AC is highlighted in a yellow rectangle, and magnified on the right with channels shown separately and merged, in which scale bar represents 2 μm. c Immunofluorescence of H3K9ac on first-generation ACs at different cell stages, and ACs that have been propagated for generations and endogenous chromosomes in GFP::LacI- and GFP::LacI::HDA-1-tethering strains. Cropped images containing ACs and endogenous chromosomes (Endo Chr.) were shown. Embryos were stained with antibody against H3K9ac (red), antibody against LacI (green) and DAPI (blue), shown separately and merged. Scale bar represents 1 μm for both ACs and endogenous chromosomes. Quantification of IF signals. Mean-corrected histone H3K9ac signal in each ROI was normalized with mean-corrected DAPI signal in each ROI, and the average normalized histone H3K9ac signal intensity was calculated. The number of cells (n) analyzed was indicated. Error bars indicate 95% confidence interval (CI) for the mean. ***p < 0.001 and **p < 0.01 by Student’s t test. NS means not significant. Black arcs show comparisons between GFP::LacI- and GFP::LacI::HDA-1-tethering strains at the same cell stage. Blue arcs show comparisons between ACs at different stages in GFP::LacI-tethering strain. Green arc shows a significant difference between 1 and 8-cell stage first-generation ACs and endogenous chromosomes in GFP::LacI-tethering strain. d Immunofluorescence of H4ac on first-generation ACs at different cell stages, and ACs that have been propagated for generations and endogenous chromosomes in GFP::LacI- and GFP::LacI::HDA-1-tethering strains. Cropped images containing ACs and endogenous chromosomes (Endo Chr.) were shown. Embryos were stained with antibody against H4ac (red), antibody against LacI (green) and DAPI (blue), shown separately and merged. Scale bar represents 1 μm for both ACs and endogenous chromosomes. Quantification of IF signals. Mean corrected histone H4ac signal in each ROI was normalized with DAPI signal in each ROI, and the mean normalized histone modification signal intensity was calculated. The number of cells (n) analyzed was indicated. Error bars indicate 95% confidence interval (CI) for the mean. ***p < 0.001 and **p < 0.01 by Student’s t test. NS means not significant. Black arcs show comparisons between GFP::LacI- and GFP::LacI::HDA-1-tethering strains at the same cell stage. Blue arcs show comparisons between ACs at different stages in GFP::LacI-tethering strain. Green arc shows a significant difference between 1 and 8-cell stage first-generation ACs and endogenous chromosomes in GFP::LacI-tethering strain

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