Stra13 is sumoylated.

<p>(A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three α-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. β-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxα, or PIASy as indicated. Lysates were immunoprecipitated with Myc- agarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS.</p

Sumoylation is essential for Stra13-dependent growth inhibition.

<p>(A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B–C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean ±SD.</p

Mutation of sumoylation sites abrogates Stra13-mediated growth suppression.

<p>(A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B–C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean ±SD. (E) Stra13^−/− MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments.</p

Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization.

<p>(A) mRNA levels of cyclin D1, p21^Cip/WAF, cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean ±SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter.</p

HDAC1 regulates Stra13 sumoylation.

<p>(A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti-Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1. TSA was added at a concentration of 300 nM. Cell lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. (C–D) NIH3T3 cells were left untreated (−) or treated (+) with TSA. Endogenous Stra13 was immunoprecipitated and analyzed for sumoylation (C), as well as for association with HDAC1 (D). (E–F) Down-regulation of endogenous HDAC1 expression in siHDAC1 cells compared to control cells was examined by western blotting (E). Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G–I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng). 48 hr later, luciferase activity was assayed. (K) siHDAC1 cells and controls were transfected with pD1luc in the absence and presence of Stra13. Luciferase activity was measured 48 hr later. Error bars indicate mean ±SD.</p

Unbiased network modeling links cortical signaling with clock components via SHARP1, BMAL1 and PER1.

<p>A) Depicted is the network model with the highest significance computed with all genes found to be differentially regulated in the cortex at ZT4 versus ZT16 (see. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone-0110310-g002" target="_blank">Figure 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone.0110310.s010" target="_blank">Tables S1</a>-S3 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone.0110310.s009" target="_blank">Figure S9</a> for description of symbols) including SHARP1 and -2. The network connects 14 seed nodes depicted as blue circles (higher expression levels in WT are indicated by associated small red circles) extended by 13 interactors. SHARP1 and -2 are labeled by red circles; all nodes added by the algorithm are not underlined by colored circles. The structure depicts two major clusters and places the circadian regulators SHARP1 and SHARP2 as well as PER1 at central positions. The left cluster (n = 19 objects) is mainly comprised of cellular signaling components (enkephalin A, substance P both encoded by <i>Penk</i> and the GPCRs A2A and DRD2) and downstream effectors including negative regulators (DUSP1,6 and HSPs) as well as transcriptional mediators (e.g. FOS, EGR1, JUNB). The right cluster (n = 12) comprises central components of the molecular clock (e.g. the core clock transcription factors CLOCK, NPAS2 and BMAL1 as well as clock feedback regulators and modifiers including SHARP1 and -2, PER1 and -2, CRY1, DBP and NR1D1/Rev-ERBalpha). The extended network gene list was queried against the GeneGo database for enriched correlations with diseases (B) and biological processes (C). Among the ten most significant disease associations were nine mental or mood related disease classifications (B). Among the ten highest ranked biological processes were only circadian rhythm- (rank 1 and 2) and metabolism-associated (rank 3–8) processes (C). MeSH ID, unique Medical Subject Heading disease identifier; GO ID, unique identifier of the gene ontology biological process collection; p-values determined by hypergeometric tests.</p

S1/2^-/- mice display novelty induced hyperactivity, decreased anxiety and exploratory behavior and working memory disturbances.

<p>A–C) Open field test performed in a novel, unfamiliar test arena. WT: n = 24, S1/2^-/-: n = 26. A) Novelty-induced hyperactivity in S1/2^-/- mice as assessed by moving distance in the open field (p_MW = 0.0006). B) Analysis in 1 min bins yielded a significant E_genotype (F_(1,48) = 16.46; p = 0.0002). Moreover, Bonferroni posttest revealed the strongest difference between the genotypes in interval 3, 5 and 10 (p_Bonf<0.01, p_Bonf<0.05 and p_Bonf<0.05, respectively). C) Mutants spent more time in the center (p_MW = 0.0004) of the test arena indicating reduced anxiety when compared to controls. D-E) Hole board test performed with a subsequent modification of the open field setup by floor insert with holes. WT: n = 24, S1/2^-/-: n = 26. D) S1/2^-/- mice displayed no alterations in the overall activity measured as total distance travelled. E) S1/2^-/- mice performed less nose pokes into holes (p_MW = 0.0014) indicating decreased curiosity-related behavior compared to WT. F-G) Y-maze test. WT: n = 23, S1/2^-/-: n = 20. F) S1/2^-/- mice showed increased activity in Y-maze test (E_genotype F_(1,41) = 10.98; p = 0.0019) most evident in interval 0-5 min (p_Bonf<0.01). G) Mutant mice performed less alterations in Y-maze than control animals (E_genotype F_(1,41) = 4.86; p = 0.0331) and p_Bonf<0.05 for interval 5–10 min. H-J) S1/2^-/- mice display impairment of working memory in the radial arm water maze (RAWM). WT: n = 29, S1/2^-/-: n = 28. H) In the visible platform task, performance was similar in both genotypes (E_genotype F_(1,55) = 0.65; p = 0.4236). I-J) S1/2^-/- mice showed increased number of working errors searching for a hidden platform on the first (I) (E_genotype F_(1,55) = 3.93; p = 0.0524; I_{genotype×time} F_(3,165) = 2.68; p = 0.0486) and the second (J) day of experiment (E_genotype F_(1,55) = 9.05; p = 0.0044) and I_{genotype×time} F_(5,275) = 2.34; p = 0.0422). Bonferroni posttest revealed significant difference during the 3^rd trial of the second day (p_Bonf<0.001). WT, black bars/circles. S1/2^-/-, white bars/circles. Data were analyzed with 2-way ANOVA with Bonferroni posttest (p_Bonf) and Mann-Whitney test (p_MW) for pairwise comparisons. ***: p<0.001; **: p<0.01; *: p<0.05. E, effect; I, interaction of factors.</p

S1/2^-/- mice show alterations of prepulse inhibition (PPI) which are resistant to Clozapine treatment.

<p>A) S1/2^-/- mice display impairment of PPI (E_genotype F_(1,43) = 7.99; p = 0.0071). Bonferroni posttest revealed significant difference in prepulse 75 und 80 dB (p_Bonf<0.01 and p_Bonf<0.05, respectively). WT: n = 24, S1/2^-/-: n = 21. B) Startle response was similar in S1/2^-/- mice and WT controls (E_genotype F_(1,88) = 0.00; p = 0.9958) and not influenced significantly by vehicle injections (E_treatment F_(1,88) = 2.18; p = 0.1434). Acute clozapine treatment (3 mg/kg) reduced startle in both genotypes to similar extend (E_treatment F_(1,82) = 11.83; p = 0.0009 and E_genotype F_(1,82) = 0.01; p = 0.9030). ‘No injections’ and ‘vehicle’ groups: WT: n = 25, S1/2^-/-: n = 21; clozapine: WT: n = 20, S1/2^-/-: n = 20. C) Acute treatment with clozapine (cloz; 3 mg/kg; n = 20) reduced PPI in WT mice when compared to vehicle (veh; n = 24) treated WT animals (E_treatment F_(1,42) = 10.33; p = 0.0025). Bonferroni posttest confirmed significant difference when prepulse 70 dB was applied (p_Bonf<0.01). D) Acute treatment with clozapine (cloz; 3 mg/kg) did not influence PPI in S1/2^-/- mice (n = 20) when compared to vehicle injected mutants (n = 21) (E_treatment F_(1,39) = 0.00; p = 0.9716). Data were analyzed with 2-way ANOVA and Bonferroni posttest (p_Bonf). ***: p<0.001; **: p<0.01; *: p<0.05. E, effect.</p

Attenuated sleep-wake amplitude and activity profiles in S1/2^-/- mice.

<p>A) Group means (± SEM) of the total time spend in different vigilance states over 24 h LD periods. The overall time of wake, NREM and REM sleep remained unaltered between genotypes. WT: n = 7, filled bars S1/2^-/-: n = 8, empty bars. B) Group means of light-dark or amplitude differences for wake, NREM and REM sleep. S1/2^-/- animals showed a significantly reduced light-dark amplitude for all vigilance states compared to WT animals (E_genotype F_{(1, 39)} = 19.87, p<0.0001; E_{vigilance state} F_{(2, 39)} = 19.39, p<0.0001; I_{genotype×vigilance state} F_{(2, 39)} = 1.9, p = 0.16; Post hoc two-tailed T-test: **: p<0.01 *: p<0.05). WT: n = 7, filled bars S1/2^-/-: n = 8, empty bars. C) 24 h sleep-wake distribution plotted for representative individual WT (#26) and S1/2^-/- (#828) mice with black areas given as relative amount of wakefulness obtained from 5 min bins. Note the relative difference in the amount of wakefulness during the light and dark episodes in the WT and the short periods of wakefulness in the light phase. In contrast, the S1/2^-/- mouse displayed broadened periods of sleep and wakefulness during the light and dark phases. D-F) Time course of vigilance states wakefulness (D), NREM (E) and REM sleep (F). Curves connect 2 h bin mean values (± SEM) expressed as percentage of recording time (E_time: NREM: F_(11,120) = 9.74, p<0.0001; REM F_(11,120) = 9.98, p<0.0001; E_genotype: NREM n.s.; REM F_(1,120) = 7.65, p<0.01 and I_{genotype×time}: Wakefulness F_(11,120) = 2.06, p = 0.02; NREM: F_(11,120) = 1.82, p = 0.05; REM: F_(11,120) = 1.42, p =  0.17; * =  p<0.05 in two-tailed post hoc T-test. WT: n = 7, filled circles S1/2^-/-: n = 8, empty circles. G) Diurnal wheel-running profiles depicted as accumulated activities of all recordings over a 5-day period plotted as 18 min bins. S1/2^-/- mice displayed a significantly altered activity profile in LD compared to wild-type (WT) mice (I_{genotype×time} F_(86,39040) = 1.92, p<0.0001) with reduced half maximal values of nocturnal activities at ZT 17.1 for S12^-/- mice compared to WT controls with ZT 18.3. Bonferroni posttest revealed significantly reduced activities between ZT13 and 18 (p_Bonf<0.05). n = 12 each genotype. H-I) Daytime dependent gene expression analysis of the circadian marker gene <i>Per2</i> (H) and the activity-induced gene <i>Fos</i> (I) in the cortex. Daytime dependent cortical expression of the circadian marker gene <i>Per2</i> was not substantially altered in WT and S1/2^-/- mice (H). In contrast, the mRNA expression of the activity regulated marker gene <i>Fos</i> was significantly reduced in S1/2^-/- mice at ZT16 compared to WT (I). n = 3 per timepoint and genotype. Data were analyzed with 2-way ANOVA with Bonferroni posttest (p_Bonf) and Mann-Whitney test (p_MW) for pairwise comparisons. E, effect; I, interaction of factors.</p

Probing p300/CBP Associated Factor (PCAF)-Dependent Pathways with a Small Molecule Inhibitor

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity <i>in vitro</i>. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation