H3.3 ends analysis profiles. (A) From chromatin isolated from intact nuclei using 80-mM salt; (B) from chromatin isolated from intact nuclei using 600-mM salt after depletion of the 80-mM salt fraction; and (C) from EDTA-treated nuclei using 350-mM salt. See the legend to Figure 5 for details.

H3.3 landscapes compared to other chromatin marks. The same gene-rich region displayed in Figure 4 is shown at lower and higher magnification. Blue tracks display H3.3 landscapes and brown tracks display landscapes based on data from previous studies: RNA polymerase II and H2A.Z (35) and H3K36me3, H3K4me3, H3K9me3 and H3 (34).

Enrichment of chromatin salt fractions and H3.3 locally within transposons. Heat-map stacks of 10 transposon families for salt fractions, mononucleosomes and H3.3. (Top row) Successive 80-mM and 600-mM salt-extracted fractions and the insoluble pellet profiled versus MNased nuclear starting material, and gel-purified mononucleosomes/input bulk DNA. (Second row) H3.3/input levels for successive 80-mM and 600-mM or 350-mM pull-down fractions and for a 350-mM extract. Vertical gray streaks result from alignment gaps.

Ends analysis profiles of salt fractions and extracts. Genes with both ends annotated were aligned at their 5′ and 3′ ends and rank-ordered by gene expression, then divided into quintiles for averaging. Means of two biological replicates are shown as log-ratios. (A) Eighty-millimolar salt fractions; (B) 600-mM salt fractions; (C) 600-mM insoluble pellet- (D) A + T content; (E) gel-excised mononucleosomes. The X-axis interval unit is 100 bp.

Profiles of chromatin obtained from salt fractions, nuclear extracts and gel-purified mononucleosomes. A typical gene-rich region of the C. elegans genome is shown with annotated (WS190) 5′-to-3′ ends shown for genes oriented left to right above the line and from right to left below. Tracks in blue represent log-ratios after triangular smoothing by averaging each probe with its neighboring probe on either side. Pairs of biological replicates are displayed in successive tracks: (A, B) 80-mM salt fractions; (C, D) 600-mM salt fractions; (E, F) 600-mM insoluble pellet; (G, H) gel-excised mononucleosomes.

The scheme for purification of biotinylated chromatin in C. elegans embryos. We first prepared a biochemical quantity of gravid adult worms expressing biotinylated BioTag::H3.3. After obtaining nuclei from worm embryos, we subjected the nuclei to micrococcal nuclease digestion in buffer A (containing ∼17 mM salt). Next, we performed centrifugation to separate the supernatant from the MNase-treated nuclei. The nuclear pellet was then resuspended and extracted in low salt buffer (80 mM), followed by centrifugation to separate 80-mM salt-soluble chromatin from the pellet. The pellet was then resuspended in high salt buffer (600 mM), followed by centrifugation to separate the 600-mM salt-soluble chromatin from the 600-mM salt-insoluble pellet. In the next step, we used the 80-mM and 600-mM salt-soluble chromatin as inputs to perform affinity pull-down with streptavidin–Sepharose. We then recovered DNA from different steps during the streptavidin pull-down procedure, including the inputs, pulled-down and unbound nucleosomes, labeled the DNA, and hybridized the labeled DNA onto micorarrays.

DNA and protein characterizations of C. elegans embryo chromatin fractions. (A) DNA recovered from different chromatin fractions was resolved on a 1.5% agarose gel and stained with ethidium bromide. MNase-digested chromatin produced a typical nucleosomal ladder (lane 3). A sufficient amount of DNA was recovered from the 350-mM salt pull-down sample, and the streptavidin–Sepharose pull-down DNA is oligonucleosomal in nature (lane 8). Under this chromatin isolation condition, we found that a portion of the mononucleosomes leaks out into the 0.1 M salt supernatant fraction (lane 4). Lanes 1 and 11: DNA molecular weight standard; lane 2: nuclei; lane 3: an MNase-treated sample; lanes 4–6: the input, streptavidin pull-down and unbound fractions of the 0.1 M supernatant, respectively; lanes 7–9: the input, streptavidin pull-down and unbound fractions of 350-mM salt-soluble samples, respectively; lane 10: 350-mM salt-insoluble pellet. (B) Protein samples from different steps of the biotinylated chromatin purification were subjected to electrophoresis on an 18% SDS–PAGE gel and probed with streptavidin-HRP and an anti-HA antibody. Biotinylated BioTag::H3.3 could be detected in the 350-mM salt-soluble streptavidin pull-down fraction, but not in the unbound lane. Lane 1: nuclei; lane 2: an MNase-treated sample; lanes 3–5: respectively the input, unbound and streptavidin pull-down fractions of 350-mM salt-soluble samples; lane 6: the 350-mM salt-insoluble pellet. (C) Biotinylated chromatin isolation and affinity purification were performed. Nuclei were resuspended in buffer A containing ∼17 mM salt for MNase digestion, and the digestion was stopped using EGTA. Next, centrifugation was performed to separate the supernatant containing MNase from the pellet containing nuclei. The nuclei were then resuspended in 80-mM salt, extracted, then the pellet was resuspended in 600-mM salt and extracted again. The 80-mM and 600-mM salt-soluble chromatin fractions, together with the 600-mM salt-washed pellet, were used for affinity purification with streptavidin–Sepharose. DNA recovered from different chromatin fractions was resolved on a 1.5% agarose gel and stained with ethidium bromide. MNase digested chromatin produced a typical nucleosomal ladder (lane 4). Under this chromatin isolation condition, no mononucleosomes leak into the supernatant during the centrifugation step performed after MNase digestion (lane 5). The 80-mM salt-soluble fraction consists of only mononucleosomes (lane 6), while the 600-mM salt-soluble fraction shows a typical nucleosomal ladder (lane 8). A low amount of DNA remains in the pellet (lane 10). Lanes 1 and 12: DNA molecular weight standards; lane 2: nuclei; lanes 3 and 4: respectively samples treated with MNase for 4 or 10 min; lane 5: supernatant; lanes 6 and 7: respectively the input and streptavidin-unbound component of the 80-mM fraction; lanes 8 and 9: respectively the input and streptavidin-unbound component of the 600-mM fraction; lanes 10 and 11: respectively the input and streptavidin-unbound fraction of 600-mM salt-washed pellet.

In vivo chromatin biotinylation in C. elegans. Diagrams of (A) BioTag::H3.3 and (B) BirA::gfp transgenes used in this study. The H3.3 transgene was fused to the BioTag and the three hemagglutinin (3XHA) epitope tag at its N-terminus. This BioTag is a ϵ-amino-acid peptide containing a lysine whose ϵ-amino group serves as substrate for biotinylation by the E. coli Biotin Ligase (BirA). A linker amino acid sequence (SRPVAT) was used to fuse BIRA to GFP. Each transgene included 1 kb upstream and 1 kb downstream of the his-72 coding sequence. (C) An adult hermaphrodite fixed and stained for HA and DNA. BioTag::H3.3 is present in the germline and somatic cells (white arrows) of adult hermaphrodites and co-localizes with DNA, except in the mature oocytes, where it is stored in the nucleus as a maternal factor. Scale bar, 50 µm. (D) GFP fluorescence and DIC images of dissected embryos expressing BIRA::GFP. GFP fluorescence could be detected starting from the 50–80-cell stage embryos, and is distributed in both the cytoplasm and nuclei of the embryo. Scale bar, 50 µm. (E) In vivo biotinyation of BioTag::H3.3 is efficient in C. elegans. Western blot analysis using Streptavidin-HRP (top panel) and an HA antibody (bottom panel) for mixed stage worm strains expressing BioTag::H3.3 alone (lane 1), GFP::BIRA alone (lane 2), BioTag::H3.3 in combination with BIRA::GFP (BioTag::H3.3/BIRA::GFP, lane 3) and OP50 Bacteria (lane 4). Biotinylation of BioTag::H3.3 (∼21.4 kDa) could be detected only in worm strains expressing both BioTag::H3.3 and BIRA::GFP. The band residing between 16 and 25 kDa in the OP50 bacteria (food for the worm used to prepare the samples) lane is the endogenous biotinylated protein in bacteria. The anti-HA western blot experiment confirmed that the band detected by streptavidin::HRP is the transgene, since they have the same migration rate in the gel. (F) BioTag::H3.3 is incorporated into chromatin. Histones were extracted with increasing salt concentrations from embryos expressing BioTag::H3.3/BIRA::GFP. Core histones elute with 2.5 M NaCl but not with 600 mM NaCl. High salt (2.5 M) extraction was used to separate core histones from chromatin-associated proteins. Coomassie blue staining (top panel) and western blot analysis using Streptavidin-HRP (middle panel) and an HA antibody (bottom panel) are shown for samples retrieved during the core histone extraction procedure. Like core histones, BioTag::H3.3 elutes primarily at 2.5 M NaCl. Lane 1, nuclei resuspended in 350 mM NaCl; lanes 2–4: respectively the first, second and third eluates of the nuclear pellet resuspended in 600 mM NaCl; and lanes 5 and 6: respectively the first and second eluates of nuclei resuspended in 2.5 M NaCl.