Fig 1: ARID1A interacts with the HDAC1-RSF1 complex to control the distribution of activating histone marks at the DSBs (A, B) Normalized ACT-seq coverage showing the enrichment of H3K27ac at HR- and NHEJ-prone DSBs, respectively, at the indicated time points in WT and ARID1A-KO cells. (C, D) Normalized ACT-seq coverage showing the enrichment of H2A118ac at HR- and NHEJ-prone DSBs, respectively, at the indicated time points in WT and ARID1A-KO cells. (E, F) Enrichment of H2AK118ac and HDAC1, respectively, at the HR-prone DSBs in WT and ARID1A-KO cells, measured by ChIP-qPCR. Data are presented as mean ± SEM, and Student's t test was performed. All data presented in this figure are from n = 3 independent experiments (biological replicates). Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.
Fig 2: OK107 and c772-mediated knockdown of Rpd3 impairs LTM.A. Flies were reared and trained at 29°C then transferred to 25°C thirty minutes prior to testing, which was also at 25°C. Raising and training flies at 29°C did not impair LTM in parental control flies, confirming that the higher temperature itself was not detrimental to LTM formation. Knockdown of Rpd3 throughout development with OK107-GAL4 resulted in a significant impairment in LTM compared to control genotypes (*p<0.05, **p<0.01) B. Flies were raised to adulthood at 19°C and switched to 29°C three days prior to testing at 25°C. The loss of LTM in the Rpd3 knockdown males demonstrates that the memory phenotype was caused by an adult-specific decrease in Rpd3 (*p<0.05, **p<0.01). C. c772-GAL4 mediated knockdown of Rpd3 abolished LTM whereas the GAL4 driver control flies had normal LTM (**p<0.01). Knockdown of Rpd3 with c739-GAL4 did not affect LTM. D. Confocal projection through the mushroom body lobes stained with FasII reveal no obvious abnormalities in lobe structure caused by c772-GAL4 knockdown of Rpd3. Scale bar = 50 µm.
Fig 3: Knockdown of Rpd3 has no impact on STM.To test the effect of OK107-GAL4 knockdown of Rpd3 on STM, flies were raised to adulthood at 19°C and switched to 29°C three days prior to testing. For evaluation of immediate recall (A), flies were trained for one hour and then tested, and for STM (B) flies were tested one hour after training. All genotypes showed normal immediate recall and one hour STM, with no difference between Rpd3 knockdown flies and parental controls.
Fig 4: FOXK2 acts as a chromatin targeting factor for the BAP1-containing deubiquitinase complex. ChIP analysis of endogenous FOXK2 and BAP1 binding to the indicated genomic loci in U2OS cells treated with non-targeting control siRNAs (siNT, black bars) or siRNA against FOXK2 (siFOXK2, white bars) (A) or siRNA against BAP1 (siBAP1, grey bars) (B). ChIP was performed with nonspecific IgG or anti-FOXK2 and BAP1 antibodies; the data are shown relative to the input DNA and are the averages of two (B) or three (A) experiments. (C) ChIP analysis of H2A and H2AK119 ubiquitin levels at the indicated genomic loci in U2OS cells treated with non-targetting control siRNAs (siNT; light grey bars), siRNA against BAP1 (siBAP1; dark grey bars), siRNA against FOXK2 (siFOXK2; white bars) or both BAP1 and FOXK2 (black bars). ChIP was performed with non-specific IgG, anti-H2A and ubiquitinated K119 H2A antibodies (H2Aub); the data are the averages of three experiments. Statistically significant differences between siNT and siBAP1/siFOXK2 treated samples are shown; P-value <0.05 (*) and <0.01(**). (D) Model depicting the role of FOXK2 in nucleating the recruitment of chromatin remodelling complexes to chromatin. FOXK2 recruits both the PR-DUB and the SIN3A complex, potentially through direct or indirect interactions with the shared subunit HCFC1. Question marks denote uncertainty about which interactions are direct. BAP1 can then cause local histone deubiquitination, and histone deacetylation can potentially be achieved through the HDAC1 component of the SIN3A complex.
Fig 5: Gawky controls nuclear import of metal-activated MTF-1. (A–D) S2 cells were treated with 500 μM copper or 50 μM cadmium for 12 h. (A, B) Proteins from nuclei (Nuc), cytoplasm (Cyto), or whole-cell extracts (Total) were subjected to western blotting analyses. HDAC1 served as a marker for the nuclear fraction. α-Tubulin served as a marker for the cytoplasmic fraction. Representative blots are shown. The level of MTF-1 protein was quantified using ImageJ from three independent western blotting experiments. The Nuc/Cyto ratio of MTF-1 was also calculated. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. (C, D) Confocal microscopy analyses of MTF-1 were performed with anti-MTF-1. Representative images are shown. Scale bars, 8 μm. n = 3. (D) Statistics of nuclear MTF-1 signal in each condition. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 40 cells for each condition. ∗∗P < 0.01; ∗P < 0.05.
Supplier Page from Abcam for Anti-HDAC1 antibody - Nuclear Loading Control and ChIP Grade