Fig 2: Related to Discussion(A) Shown are normalized density profiles of Pol II ChIP-seq (red), GRO-seq (black), RNA-seq (brown), H3K9me3 ChIP-seq (green) and piRNA-seq (light green) for the indicated OSC knockdowns (left). Shown is the ~20kb area around the transcriptional start site of the flamenco cluster. Shown are only reads mapping uniquely to the genome but we note that nearly all areas in this window are genome-unique.(B) Western blot showing protein levels of Armi, Piwi, Lamin, Mael, HP1 and Histone 3 (H3) in cytoplasmic, nucleoplasmic, soluble and insoluble chromatin fractions of OSCs. The relative amount of each fraction loaded per lane (based on fraction volume) is given below. The following antibodies were used: a-Lamin (ADL67.10, DSHB), a-HP1 (C1A9, DSHB) and a-H3 (Abcam, ab1791).

Fig 3: Kcr is reduced at DNA damage sites in an HDAC-dependent manner. (A and B) Representative images of U2OS cells that were exposed to laser microirradiation and 5 min later fixed and costained using the following antibodies: pan crotonyllysine (PTM-BIO; PTM-501, Zhejiang, China), crotonylated H3K9 (PTM-BIO; PTM-516), and ?H2AX (Cell Signaling; 2577, Danvers, MA, USA). DNA was stained with DAPI (blue). Results are typical of four independent experiments (n > 50). The percentage of cells showing colocalization of the indicated markers is written on the right. Scale bar is equal to 2 µm. (C–E) show H3K9cr levels at different time points after DNA damage induced by IR (10 Gys; C), UV (10 J/m2; D), and VP16 (40 µm; 30 min; E). Histones were prepared by acidic extraction and subjected to western blot analysis. Histone H3 (Abcam; ab1791, Cambridge, MA, USA) is used as a loading control. ?H2AX is used as a marker for DNA damage induction. The numbers below the blots indicate the ratio between the intensities of H3K9cr and total H3 bands, which was normalized to the untreated samples and averaged from three independent experiments. Band quantification was performed using ImageJ software. (F) Western blot shows the levels of H3K9cr after treatment with 5 mM NAM for 4 h (Sigma; N0636, Rehovot, Israel) or 1 µm TSA (Sigma; T1952) for 2 h. (G) as in E except for pretreating the cells either with DMSO or NAM prior to VP16 treatment. (H and I) as in D and E except for pretreating the cells either with DMSO or TSA prior to DNA damage induction. The two antibodies used in this study, pan crotonyllysine (PTM-501) and crotonylated H3K9 (PTM-516), have been tested for their selectivity and specificity by at least two independent groups (Tan et al., 2011; Andrews et al., 2016). Bands quantification was performed as described above.

Fig 4: Elevation of the levels of histone H3 as well as histone H3 acetylation at the core promoters of GAL1, ADH1 and PHO84 in ?sgf73. (A) Analysis of the levels of histone H3 acetylation at the promoters of GAL1, ADH1, PHO84 and RPS5 in the SGF73 deletion mutant strain. The wild-type and SGF73 deletion mutant strains were grown, crosslinked and immunoprecipitated as in Figure 4A. The percentages of DNA immunoprecipitated in ?sgf73 relative to wild-type (%WT) at the core promoters of GAL1, ADH1, PHO84 and RPS5 are plotted in the form of a histogram. (B) Analysis of the levels of histone H3 at the promoters of GAL1, ADH1, PHO84 and RPS5 in the SGF73 deletion mutant strain. The wild-type and SGF73 deletion mutant strains were grown and crosslinked as in panel A. IP was performed using an anti-histone H3 antibody (Ab1791; Abcam) and a modified ChIP protocol as described in Materials and Methods section.

Fig 5: The prenucleosome, a conformational isomer of the nucleosome. (A) p300 specifically acetylates histone H3K56 in prenucleosomes relative to nucleosomes. Chromatin assembly reactions with ACF (Fyodorov and Kadonaga 2003; Torigoe et al. 2011) were performed with relaxed circular plasmid DNA in the presence of acetyl-CoA. ATP (or UTP as the -ATP control), DNA, and p300 were included as indicated. In addition, as a test for acetylation at H3K56, we performed parallel reactions with the mutant histone H3K56A, which cannot be acetylated at H3 residue 56. The resulting samples were then subjected to Western blot analysis with H3K56ac-specific antibodies (Millipore, catalog no. 07-677). As a reference, the blot was stripped and reprobed with anti-total H3 antibodies (Abcam, catalog no. AB1791). (B) Prenucleosomes comprise a core histone octamer and 80 bp of DNA at a location that is analogous to that of the central 80 bp of the core particle. H3K56 is accessible to p300 in a prenucleosome but not a nucleosome. Prenucleosomes can be converted into canonical nucleosomes by a motor protein such as ACF or Chd1. (C) Prenucleosomes or prenucleosome-related particles may be present in the upstream region of active promoters. (D) Model for the productive dynamic interconversion between prenucleosomes and nucleosomes. Prenucleosomes can be formed by the deposition of histones onto DNA and converted into nucleosomes by an ATP-driven motor protein such as ACF or Chd1. Nucleosomes can be disrupted by the action of enzymes such as polymerases as well as some ATP-driven chromatin remodeling factors. The resulting free histones are bound by the chaperones and then reassembled into prenucleosomes. It is not known whether a canonical nucleosome can be directly converted into a prenucleosome.