dRYBP binds to ubiquitin and ubiquitylated proteins.

<p>(<b>A</b>) Phylogenetic conservation of the dRYBP protein consensus NZF domain. Indicated are the aminoacids required for Zn binding (orange), for protein folding (blue) and for Ub binding (arrows, green). (<b>B</b>) Western Blot of immunoprecipitation using α-dRYBP antibody of <i>Drosophila</i> embryonic nuclear protein extracts for dRYBP and Ub detection. Input: <i>Drosophila</i> embryonic nuclear protein extracts. Mock: pre-immune serum. Indicated are the 17 kDa and 25 kDa bands corresponding to dRYBP and dRYBPub. (<b>C</b>) Western Blot detection with α-Ub antibody of dRYBP-GST or dRYBPΔNZF-GST-pulldowns from S2 cell extracts treated with proteasome inhibitors (+MG-132+Lact.). Input: S2 cell extracts. Mock: protein-GST.</p

<i>dRYBP</i> interacts genetically with <i>Sce</i>, <i>dkdm2</i> and <i>dBre1</i>.

<p>(<b>A</b>) Wild type male legs (L1, L2 and L3). L1 presents the sex comb (arrowhead) not present in L2 or L3. (<b>B</b>) <i>dRYBP¹/dRYBP¹; Pc³/dkdm2^KG04325</i> legs showing ectopic sex combs on L2 and L3 (arrowheads). (<b>C</b>) Wild type wing. (<b>D</b>) <i>dRYBP¹/dRYBP¹; Pc³/dkdm2^KG04325</i> wing partially transformed to haltere. Scale bars represent 200 µm. (<b>E</b>) Wild type male abdomen. Indicated are the A4, A5 and A6 segments. Note the pigmentation of A5 and A6. (<b>F</b>) <i>dRYBP¹/dRYBP¹; Pc³/dkdm2^KG04325</i> male abdomen. Note the patches of pigmentation (arrowheads) in A4. (<b>G</b>) <i>dRYBP¹/dRYBP¹; trx^E2</i>/+ male abdomen showing patches of de-pigmentation (arrowhead) in the A5. (<b>H-K</b>) Graphs representing the frequency of the indicated phenotypes in flies of the indicated genotypes. (<b>H</b>) Genetic interaction between <i>dRYBP</i> and <i>dkdm2</i> in a <i>Pc</i> mutant background. Arrows mark the frequency of the indicated phenotypes in <i>dRYBP¹/dRYBP¹</i>; <i>dkdm2^KG04325/Pc³</i> flies (n = 100 in all cases). (<b>I</b>) Genetic interaction between <i>dRYBP</i> and <i>dkdm2</i> in a <i>trx</i> mutant background. Arrows mark the frequency of the indicated phenotypes in <i>dRYBP¹/dRYBP¹</i>; <i>dkdm2^KG04325/trx^E2</i> flies (n = 100 for all genotypes). <b>(J</b>) Genetic interaction between <i>dRYBP</i>, <i>dkdm2</i> and <i>Sce</i> (n = 100 for <i>dRYBP¹/dRYBP¹</i>; <i>dkdm2^KG04325/</i>+ and <i>dRYBP¹/dRYBP¹</i>; <i>Sce¹/+</i>; n = 45 for <i>Sce¹/dkdm2^KG04325</i>; n = 70 for <i>dRYBP¹/dRYBP¹</i>; <i>Sce¹/dkdm2^KG04325</i>). Arrows mark the frequency of the <i>dRYBP¹/dRYBP¹</i>; <i>Sce¹/dkdm2^KG04325</i>. (<b>K</b>) Genetic interaction between <i>dRYBP</i> and <i>dBre1</i>. Arrow marks the frequency of <i>dRYBP¹/dRYBP¹</i>; <i>dBre1^kim1/dBre1^kim1</i> (n = 100 in all cases).</p

Phosphorylation of ASF1 hampers proteasome-dependent degradation.

<p>(<b>A</b>) hASF1 and dASF1 wild type or mutant proteins were expressed in HEK293T and S2 cells respectively. Protein synthesis was blocked by cycloheximide in cells incubated with (red curves) or without (black curves) proteasome inhibitors lactacistin and MG132. ASF1 protein levels were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008328#pone-0008328-g003" target="_blank">Figure 3</a> by immunoblotting for hASF1 (upper panels) and dASF1 (lower panel) every hour from 0 to 6 h and normalized to actin. All experiments were repeated at least three times and error bars show SEM. (<b>B</b>) HA-hASF1 and HA-dASF1 proteins or GFP were expressed in HEK293T cells. Cells were either treated (+Pr.Inh.) or not (-Pr.Inh.) with proteasome inhibitors lactacistine and MG132. Whole cell extracts were incubated with anti-HA beads and pulled-down proteins were analyzed on a western blot with anti-ubiquitin antibody. High-molecular weight smear represents poly-ubiquitinated ASF1. (<b>C</b>) Degradation of endogenous dASF1 dependents only partially on proteasome pathway. Protein synthesis was blocked by cycloheximide in S2 cells incubated with (red curves) or without (black curves) proteasome inhibitors lactacistin and MG132. Samples were collected every hour from 0 to 6 h. dASF1 protein was immunoblotted and quantified as above. Data are represented as mean of three experiments +/− SEM.</p

Hypothetical model for dRYBP function.

<p>(<b>A</b>) The dRRK complex (dRYBP + dRING + dKDM2) counteracts dRAF-mediated repression by increasing H3K36me2 levels and perhaps, decreasing H2Aub due to the absence of PSC. (<b>B</b>) The dRB complex (dRYBP + dBRE1) counteracts dBRE1-mediated activation by lowering H2Bub levels.</p

dRYBP inactivation modulates levels of histone modifications.

<p>Western Blot analysis of whole S2 cells histone extracts or protein extracts to analyze either levels of histone modifications or protein levels to control dsRNA-inactivation efficiency with the indicated antibodies. The reduction (%) of expression was calculated measuring and quantifying the intensity of the bands using Fiji imaging software and Tubulin expression as a reference. (<b>A</b>) Inactivation of dRYBP. Note the slight decrease of H3K36me2 and strong decrease of both H3K4me and H2Aub and the efficiency of the dRYBP inactivation (98%). (<b>B</b>) Levels of H3K27me3 in the indicated KDs. Note that H3K27me3 levels do not change and the efficiency of the inactivation (dRYBP 78%, SCE: 66%, dKDM2: 90%, PC: 56%). (<b>C</b>) Levels of the indicated proteins in the corresponding KDs. Efficiency of dRYBP reduction in dRYBP KD: 97%; in dRYBP + SCE KD: 98%; in dRYBP + dKDM2 KD: 100%; in dRYBP + PC KD: 100%. Efficiency of SCE reduction in SCE KD: 66%; in dRYBP + SCE KD: 62%. Efficiency of dKDM2 reduction in dKDM2 KD: 93%; in dRYBP + dKDM2 KD: 98%. Efficiency of PC reduction in PC KD: 62%; in dRYBP + PC KD: 68%. (<b>D</b>) Levels of H2Aub in the indicated KDs. The intensity of the bands corresponding to H2Aub and H2A was measured and quantified using Fiji imaging software calculating the different H2Aub/H2A ratios. H2Aub levels reduction (%) in dRYBP KD: 65%, in SCE KD: 95%, in dKDM2 KD: 56%, in PC KD: 5%, in dRYBP + SCE KD: 90%, in dRYBP + dKDM2 KD: 49%, in dRYBP + PC KD: 46%. Note that the decrease in H2Aub levels in dRYBP KD shown in (A) and (D) seem to be slightly different probably due to experimental conditions. Also note the efficiency of the indicated dsRNA-inactivation shown in (C). (<b>E</b>) Levels of H3K36me2 in the indicated KDs. Note levels of H3K36me2 decrease in dRYBP + dKDM2 KD in comparison to dKDM2 KD and the efficiency of the indicated dsRNA-inactivation shown in (C). (<b>F</b>) Levels of H2Bub in the indicated KDs. Note that levels of H2Bub increase in dRYBP + dBRE1 KD in comparison to dBRE1 KD and the efficiency of the indicated dsRNA-inactivation. dRYBP reduction in dRYBP KD: 93%; in dRYBP + dBRE1 KD: 98%. dBRE1 reduction in dBRE1 KD: 97%; in dRYBP + dBRE1 KD: 100%. Arrowheads indicate the bands corresponding to SCE. Arrows indicate the bands corresponding to dKDM2. Tubulin, H3, H2A and H2B were used as loading controls. Mock is GFP-dsRNA in all cases. To prove reproducibility of the results between 4–5 replicates of all the experiments were performed.</p

Mutations in TLK phosphorylation sites affect ASF1 protein levels.

<p>(<b>A</b>) Mutated dASF1 proteins fused to HA-tag were co-expressed with wild-type bio-tagged dASF1 in S2 cells and revealed by immunoblotting. Mutated Serines within dASF1 protein are indicated. (<b>B</b>) Levels of mutant HA-dASF1 proteins were quantified and normalized to bio-dASF1wt protein level and compared to the same ratio obtained for HA-dASF1wt. The graph shows the mean for three experiments and error bars show standard errors of the mean (SEM). Mutated Serines are indicated (S-A mutations – blue bars, S-D – yellow bars). (<b>C, D</b>) Mutated HA-hASF1 proteins were co-expressed with GFP in HEK293T cells. Representative immunoblots are shown (<b>C</b>) and HA-hASF1 protein levels were analyzed as above (<b>D</b>) using GFP levels as a reference. The graph shows the mean for three experiments and error bars show SEM. Mutated Serines are indicated (S-A mutations – blue bars, S-D – yellow bars).</p

dRYBP interacts biochemically with SCE, dKDM2 and dBRE1.

<p>(<b>A</b>) Mass spectrometric parameters of the dRYBP, SCE, dKDM2 and dBRE1 proteins using <i>Drosophila</i> embryonic nuclear protein extracts and dRYBP #194 and dRYBP #195 serum antibodies. Molecular weight (kDa), mascot score (MS), number of unique peptides (UP), total number of peptides (TP) identified and sequence coverage (%). (<b>B</b>) Western Blot of co-immunoprecipitations using α-dRYBP, α-SCE or α-dKDM2 antibodies for dRYBP, SCE, dKDM2, PSC, PC, PH and E(Z) detection. (<b>C</b>) Western Blot of co-immunoprecipitations using α-dRYBP or α-dBRE1 antibodies for dRYBP, dBRE1, SCE, dKDM2 and E(Z) detection. In (B) and (C) <i>Drosophila</i> embryonic nuclear protein extracts (Input), pre-immune serum (Mock). (<b>D</b>) Western Blot detection with the indicated antibodies of dRYBP-GST or dRYBPΔNZF-GST-pulldowns using <i>Drosophila</i> embryonic nuclear protein extracts (Input) or protein-GST (Mock). Arrowheads point to the corresponding protein bands detected with the indicated antibodies (other bands may be non-specific or correspond to modified proteins).</p

TLK activity is required for ASF1 stability <i>in vivo</i>.

<p>(<b>A</b>) Depletion of dTLK from Drosophila S2 cells leads to decreased dASF1 levels. S2 cells were either mock treated or incubated with dsRNA directed against dTLK. Whole-cell extracts were prepared and analyzed by Western blotting with indicated antibodies. Actin serves as a loading control. (<b>B</b>) siRNA for hTLK1 and hTLK2 affects hASF1a stability in HeLa cells. Whole-cell extracts from control or siRNA treated cells were analyzed by Western blotting with anti-hASF1a and anti-Actin antibodies. Efficiency of siRNA of hTLK1 (blue) and hTLK2 (yellow) was confirmed by RT-qPCR normalized to control siRNA (right panel).</p

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.

<p>(<b>A</b>) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (<b>B–C</b>) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (<b>B</b>) or SMC1 (<b>C</b>) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen-1003719-g004" target="_blank">Figure 4B–C</a>. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (<b>D</b>) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>E</b>) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (<b>D</b>), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s005" target="_blank">Figure S5A</a>). (<b>F</b>) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (<b>G</b>) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (<b>D</b>). Similar results were obtained for SMC1 IPs from colhicine-treated cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s006" target="_blank">Figure S6</a>). (<b>H</b>) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (<b>I</b>) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion.</p

NAP1 and PP2A act antagonistically in cohesin cycle.

<p>(<b>A</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after knockdown of NAP1, PP2A or both factors. We quantified the frequency of resolved (blue), unresolved (red) sister chromatids and loss of centromeric cohesion (Cen. Loss; green). Concomitant depletion of NAP1 and PP2A resulted in a statistically significant increase of the frequency of resolved chromatids compared to the NAP1 knockdown, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7A</a>. (<b>B</b>) Representative example of mitotic chromosomes from colhicine-treated S2 cells depleted for NAP1, PP2A or for both proteins. DNA visualized by DAPI staining is shown in red. Centromers are indicated by arrowheads, whereas loss of centromeric cohesion is indicated by full arrows. (<b>B′</b>) The localization of SA (green) on mitotic chromosomes same as in (<b>B</b>) was determined by indirect immunofluorescence. (<b>C</b>) RAD21 (green) localization on mitotic chromosomes. (<b>D</b>) MeiS332 (green) localization on mitotic chromosomes. (<b>E</b>) Depletion of PP2A restores SA phosphorylation in cells lacking NAP1. Western blot analysis of SA IPed from either mock-treated S2 cells or after knockdown (KD) of NAP1, PP2A or both proteins under normal (top panel) or denaturing (middle panel, (d)IP) conditions from asynchronously dividing cells (− colhicine) or colhicine treated cells (bottom panel, + colhicine). Blots were probed with antibodies against SA, phosphorylated serine, PP2A or NAP1. After NAP1 knockdown, SA phosphorylation levels drop substantially. Whereas depletion of PP2A alone does not affect bulk SA phosphorylation, concomitant knockdown of PP2A and NAP1 neutralized the effect of NAP1 depletion, leading to restored levels of phosphorylated SA. Antibodies against phosphoSer recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>F</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after over-expression (OE) of GFP (Mock), NAP1, PP2A, both NAP1 and PP2A or the catalytic mutant PP2A^H59Q. Quantification of mitotic phenotypes was as described above (A). Overexpression of PP2A, but not PP2A^H59Q, resulted in significant increase of the frequency of unresolved chromatids. The PP2A over-expression phenotype was rescued by co-expression of NAP1, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see . Representative examples of mitotic chromosomes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7C</a>–D.</p