Fig 1: TNKS1 is required for muscle cell differentiation. (A) Total cell extracts were prepared from exponential and differentiating C2C12 myoblasts (Exp, Day 0, Day 1, Day 2, Day 3). (Top panel) Western blot experiments were performed using antibodies against pADPr or α-tubulin (loading control). (Bottom panel) Histogram representation of the quantification of the western blot in top panel. Values were quantified using ImageJ and normalized to tubulin. (B) Phase contrast images were taken of C2C12 cells treated with scrambled control (siCTRL), or specific siRNAs against PARP1, PARP2, PARP3, PARP4, TNKS1 or TNKS2 three days after induction of differentiation. Images of a single field are shown and represent three independent experiments (scale bars, 100 μm). (C) Immunofluorescence experiments demonstrating the differentiation of C2C12 myoblasts treated with the siRNAs described in (B). (Top panel) Cells were fixed three days post-induction of differentiation, and antibodies against known markers of muscle fibers (anti-MyHC, anti-myoglobin) were used for immunostaining. DAPI was used to stain nuclei. Images of a single field are shown and represent three independent experiments (scale bars, 20 μm). (Bottom panel) Quantification of the fusion index for the C2C12 cells described in top panel. The fusion index was calculated as the ratio of the nuclei number in myotubes versus the total number of nuclei. (D) (Top panel) Immunofluorescence experiments demonstrating the differentiation of primary myoblasts treated with siCTRL or siTNKS1. Cells were fixed 3 days post-induction of differentiation, and antibodies against known markers of muscle fibers (anti-MyHC, anti-myoglobin) were used for immunostaining. DAPI was used to stain nuclei. Images of a single field are shown and represent three independent experiments (scale bars, 20 μm). (Bottom panel) Quantification of the fusion index for the C2C12 cells described in top panel. The fusion index was calculated as the ratio of the nuclei number in myotubes versus the total number of nuclei. (E, F) Total extracts were prepared from C2C12 cells transfected with scrambled control (siCTRL) or siRNA against TNKS1 (siTNKS1) and collected from exponentially growing (EXP) and differentiating C2C12 myoblasts (Day 0 and Day 2). (E) The extracts were used in western blot analysis using antibodies against pADPr or α-tubulin (loading control). (F) Histogram representation of the quantification of the western blot in the left panel. Values were quantified using ImageJ, normalized to tubulin, and shown relative to the EXP siCTRL treated condition. (G, H) Total extracts were prepared from C2C12 cells transfected with scrambled control (siCTRL) or a combination of siRNA targeting PARP1 and PARP2 (siPARP1/2) and collected from exponentially growing (EXP) and differentiating C2C12 myoblasts (Day 0 and Day 2). (G) The extracts were subjected to western blot analysis using antibodies against pADPr or α-tubulin (loading control). (H) Histogram representation of the quantification of the western blot in the left panel. Values were quantified using ImageJ, normalized to tubulin, and shown relative to the EXP siCTRL treated condition. (I) (Top panel) Immunofluorescence images of C2C12 transfected with scrambled control (siCTRL) or a combination of siRNA against PARP1 and PARP2 (siPARP1/2) Cells were fixed three days post-induction of differentiation, and antibodies against known markers of muscle fibers (anti-MyHC, anti-myoglobin) were used for immunostaining. DAPI was used to stain nuclei. Images of a single field are shown and represent three independent experiments (scale bars, 20 μm). (Bottom panel) Quantification of the fusion index for the C2C12 cells in top panel. The fusion index was calculated as the ratio of the nuclei number in myotubes versus the total number of nuclei. Data shown in Figure 1 are presented ± the s.e.m. of three independent experiments with *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test.
Fig 2: TNKS1 PARylates promyogenic RNA-Binding Proteins such as HuR. (A) Total cell extracts isolated from differentiating C2C12 myoblasts (Day 2) were used for immunoprecipitation experiments with pADPr or IgG antibodies and analyzed by mass spectrometry. The immunoprecipitation of pADPr was validated by western blot analysis using a pADPr antibody. The blot shown is representative of three independent experiments. (B) The identified proteins were classified according to their gene ontology molecular function. The percentage of proteins identified in each category is represented in a pie chart. (Lower panel) List of top 5 categories. (C) List of RNA-binding proteins previously associated with myogenesis identified in (B). (D) Immunoprecipitation experiments using pADPr or IgG antibodies were performed with extracts from confluent (D0) C2C12 cells. The association of RNA-binding proteins such as HuR and KSRP to pADPr was determined by western blot analysis. The blot shown is representative of three independent experiments. (E, F) Immunoprecipitation experiments using pADPr or IgG antibodies were performed with extracts from confluent (D0) C2C12 cell transfected with scrambled control (siCTRL) or siRNA against TNKS1 (siTNKS1). (E) The association of HuR to pADPr was determined by western blot analysis. The blot shown is representative of three independent experiments. (F) Histogram representation of the quantification of the western blot in the (E). (G) Schematic representation of the in vitro PARylation assay procedure. GST-HuR and biotinylated NAD+ were incubated with recombinant PARP1, PARP3, or TNKS1 enzymes. HRP-conjugated streptavidin was added to the reaction and the signal was measured as arbitrary units by chemiluminescence by detection with luminol. (H) Quantification of Ribosylation units demonstrating the PARylation of GST-HuR by PARP1 and TNKS1, but not PARP3, in vitro. Levels are normalized to GST control. Data shown in Figure 2 are presented ± the s.e.m. of three independent experiments with *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test
Fig 3: The Binding of PARylated PARP1 to the Macro Domain of ALC1 Triggers a Major Conformational Change(A) Difference plot of HDX data from ALC1fl in the presence and absence of PARylated PARP1. Experimental error is shown in gray, and areas of increased exposure and protection are shaded in red and blue, respectively. Different line colors show different D2O incubation times. Non-covered sequence (Figure S5C) is omitted.(B) Comparison of intra-ALC1 cross-links in non-activated or activated ALC1fl. Gray, green, and magenta lines indicate cross-links that are detected in both cases, only with activated, or only with non-activated ALC1fl, respectively.(C) SAXS-based ab initio and rigid-body models of ALC1cat.(D) The ATPase of SNF2 (Liu et al., 2017) aligned onto that of ALC1cat (cartoon) and of ALC1fl-MC (lines), using the N-lobe as a guide (not shown for SNF2).(E) SAXS-derived pairwise interatomic distance distribution for ALC1cat in the apo (orange) and DNA-bound (green) state, scaled such that the area under the curve matches the observed particle volume.See also Figure S5.
Fig 4: Mutations that Destabilize ATPase Motor-Macro Domain Interactions Constitutively Activate ALC1 and Alter the Dynamics of Its Recruitment at DNA Damage Sites(A) ATPase activities of ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown).(B) Remodeling by ALC1fl WT (same as in Figure 1C) compared to R857Q (left), R857E (middle), and R860W (right) in the presence or absence of PARP1/NAD+. Bar graph: relative rates for ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown).(C) Representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at the indicated early time points following laser damage.(D) Kinetics of YFP-ALC1 (left) and YFP-ALC1 R860W (right) association with DNA breaks as quantified from (C).(E) Half-hour time course with representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at indicated time points following laser damage.(F) Kinetics of WT, R857Q, R857E, and R860W YFP-ALC1 association with/dissociation from DNA breaks as quantified from (E).(G) Kinetics of YFP-ALC1 R857Q and R857E association with DNA breaks.(H) Kinetics of WT YFP-ALC1 dissociation from DNA breaks.Error bars ± SEM; τ: time constant; scale bars, 10 μm. See also Figure S7.
Fig 5: Catalytic Activities of ALC1fl and ALC1cat(A) Left: domain organization of ALC1fl and ALC1cat. Right: ATPase activities of ALC1fl (teal) and ALC1cat (orange).(B) Nucleosome remodeling by ALC1fl and ALC1cat. The intrinsic HhaI cut site is initially protected by the histone octamer but becomes accessible upon ALC1-induced remodeling.(C) Time courses for remodeling by ALC1fl and ALC1cat in the presence or absence of PARP1/NAD+. Bar graph: relative remodeling rates for ALC1fl (teal) and ALC1cat (orange).Error bars ± SEM. See also Figure S1.
Supplier Page from Enzo Life Sciences, Inc. for PARP-1 (human), (recombinant) (high purity)