Cell culture and cell lysate preparation
HEK293T cells were grown in DMEM (AL007A, Himedia) supplemented with 10% FBS (RM1112, Himedia) and antibiotic-antimycotic agent (15240062, Gibco). Cells were collected and washed with PBS (TL1006, Himedia). Washed cell pellet was lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA , 50 mM Tris, 1% NP40 (IGEPAL), 0.1% Na-deoxycholate and 0.1% SDS) containing Halt protease and phosphatase inhibitor cocktail (PI78441, Invitrogen).
Crosslinking of DNA with membrane disc
Genomic DNA was isolated from HEK293T cells [48]. DNA was sonicated using a Diagenode Bioruptor sonicator for 21 cycles at 30 seconds “ON” followed by 30 seconds “OFF”. Sonication was standardised to get ~1 kb fragment size DNA. Sonicated genomic DNA was dissolved in 1x TE buffer at 200 ng/µl. Nylon-membrane (GX222020NN, Genetix) discs (5 mm diameter) were soaked with the sonicated DNA overnight and were crosslinked by vacuum heating the wet membranes at 80°C for 90 minutes. DNA crosslinked nylon-membrane discs (called dot blots) were transferred to 96 well plates and incubated with blocking solution (PBST with 10% FBS). For the primary screen, one dot blot was used for each of the 1685 compounds (L1100, Selleckchem) distributed across multiple 96 well plates. In addition, the control samples, as described below, were run on two dot blots for each 96 well plate.
Dot-blot assay
DNA-crosslinked nylon membranes were pre-incubated with blocking solution for 90 minutes. Cell lysate (15 μl of a 25 ml lysate stock obtained from approximately 500 million cells) was incubated with compounds (pre-dissolved in DMSO and diluted to a final concentration of 100 μM) or compound diluent (0.05% DMSO in 1x PBS) for 30 minutes. The concentration of 100 μM for the chemicals was selected with the following considerations: The Cmax of cancer chemotherapeutic agents (mostly inhibitors of enzymatic activities) has a wide range (35.04±145.66 μM) with a median of 1.18 μM. The median Cmax of 1 μM for approximately 0.5-1 million cells in 10 ml of culture medium translates into 20-10 nmoles of chemical per 1 million cells. We extracted 500 million cells into 25 ml lysate and used 15 µl of this extract with 100 µM of each chemical in a 30 µl reaction volume. Thus, the 100 µM concentration translated into 20-10 nmoles of chemical for every million cells. This also approximated the median Cmax of 1 μM. The chosen concentration was thus optimal only for the in vitro DBID assay only. Pre-incubated compound-lysate mix was transferred to DNA-crosslinked nylon membrane and incubated for 90 minutes at 4°C. The dot blots were washed three times with 1x PBS and protein-DNA complexes were crosslinked with 4% formaldehyde in 1x PBS. The dot blots were washed three times with PBST and then incubated with 50 μl of rabbit polyclonal anti-CGGBP1 antibody mix (a mix of 1:120 dilutions of SC-292517, SCBT and 10716-1-AP, Proteintech) overnight with gentle rocking. Dot blots were washed with PBST three times and incubated with 30 μl of the biotinylated anti-rabbit secondary antibody for 90 minutes (1:10 dilution, 865002, R&D Systems). After three PBST washes of five minutes each, the dot blots were incubated with high sensitivity streptavidin conjugated to horseradish peroxidase (1:10 dilution, 865006, R&D Systems) for 90 minutes. All antibody dilutions and streptavidin-HRP conjugate dilutions were done in the blocking solution. Dot blots were washed three times with PBST and incubated with 15 μl of DAB (3,3’-Diaminobenzidine) chromogen (2 ml of DAB chromogen (860001, R&D Systems) diluted in DAB chromogen buffer (860005, R&D Systems)). Positive and negative controls are described below.
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Cell lysate
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Primary antibody
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Secondary antibody (biotinylated anti-rabbit IgG)
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Positive control
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Yes
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Anti-CGGBP1 rabbit polyclonal IgG
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Yes
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Negative control 1
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No
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Anti-CGGBP1 rabbit polyclonal IgG
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Yes
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Negative control 2
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Yes
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No
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Yes
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Negative control 3
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Yes
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IgG
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Yes
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Negative control 4
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Yes
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Anti-CGGBP1 rabbit polyclonal IgG
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No
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Secondary screening for direct or indirect inhibition of the primary hits
The secondary screening was performed for eight inhibitors obtained as hits from the primary screen along with a positive control (no inhibitor) and a negative control (no primary antibody). The assay was done in 6-well plates with each inhibitor-rCGGBP1 combination assayed in 8-10 technical replicates. For each sample, dot blots were incubated with 250 μl of blocking solution (10% FBS in PBS) for 1 hr at room temperature in a moist chamber. Simultaneously, the rCGGBP1 (500 ng per compound) diluted in RIPA lysis buffer (containing protease phosphatase inhibitor cocktail) was incubated with inhibitors (at a final concentration of 100 μM for 45 minutes) at 4°C. In the positive control sample, the rCGGBP1 diluted in RIPA lysis buffer was incubated with the compound diluent (0.05% DMSO in 1x PBS). The protein-inhibitor mix was diluted in the blocking solution such that the final volume was 250 μl for each sample. The blocking solution was removed and rCGGBP1-inhibitor mix was transferred onto the membranes in each well for 1 hr at 4°C in a moist chamber with gentle rocking. The blots were washed thrice with PBS. This was followed by fixation of the interactions using 1% PFA for 5 minutes and subsequent PBST washes three times. Membranes were incubated with mouse anti-FLAG antibody (1:1000 of SC-166384, SCBT, in blocking solution) for 1 hr at room temperature with gentle rocking, followed by washing three times with PBST. The membranes were then incubated with anti-mouse HRP-conjugated secondary antibody (1:5000 of NA931, GE Healthcare) for 1 hr at room temperature followed by three washes with PBST. The signal was detected with ECL substrate (32106, Pierce) by using Biorad ChemiDoc MP Imaging System. Further, the membranes were stripped with 0.2 N NaOH for 5 minutes and incubated with anti-CGGBP1 antibody (1:1000 of 10716-1-AP, Proteintech, in blocking solution) overnight at 4°C followed by three PBST washes. The membranes were incubated with anti-rabbit HRP-conjugated secondary antibody (1:5000 of NA934, GE Healthcare) for 1 hr at room temperature and subsequently washed with PBST thrice. The signal was captured detecting chemiluminescence as described above. The signal was quantified by densitometry analysis of images (.tif format) using ImageJ software. The statistical analysis and data presentation were performed using OpenOffice and GraphPad Prism8. The DBID assay was also performed for the Givinostat-CGGBP1 combination using a lower concentration range (10 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM) of Givinostat. This assay was repeated to detect the inhibition of DNA-binding of total histone H3 (without fixation) and histone H3 post-translational modifications: H3K9me3 and H3K4me3 (with fixation) by Givinostat.
Synthesis of Alu DNA
For the generation of full-length Alu DNA, an established DNA-binding target of CGGBP1, the full-length consensus sequence of Alu SINE was synthesized in five overlapping oligonucleotides. The 5' end of the first fragment and the 3' end of the last fragment contained the T7 and SP6 primers respectively. The Alu DNA product was obtained through overlapping PCR using an equimolar mix of overlapping oligonucleotides as template and T7 and SP6 sequences as primers. The PCR product was run on the agarose gel and purified using the PCR purification kit (A1222, Promega). The Alu PCR product was cloned into using the pGEM-T Easy Vector (A1380, Promega). The clones obtained were subjected to Sanger sequencing for verification. This clone was used as a template for amplifying Alu DNA for in vitro DNA-rCGGBP1 immunoprecipitation assay. Two different lengths of Alu DNA were amplified due to two priming sites for T7 as well as SP6 in the clone (~320 bp (T7 and SP6 sites in the insert) and other at ~400 bp (T7 and SP6 sites in the vector backbone)). The Alu DNA was amplified using either unmethylated cytosine or 5'-methylated dCTPs. The sequence of the Alu DNA is as follows:
5’-TAATACGACTCACTATAGGGGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCAC TTTGGGAGGCCGAGGCGGGAGGATCGCTTGAGCCCAGGAGTTCGAGACCAGCCTGGGCAACATAGCGAGACCCCGTCTCTACAAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGGATCGCTTGAGCCCAGGAGTTCGAGGCTGCAGTGAGCTATGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACCCTGTCTCTTCTATAGTGTCACCTAAAT-3'
In vitro DNA-IP and qPCR
The in vitro DNA-IP was performed with or without Givinostat. rCGGBP1 (0.5 μg) diluted in 1x PBS was incubated with an inhibitor (compound was pre-dissolved in DMSO and diluted to 100 μM final concentration) for 45 minutes at room temperature. The final volume was adjusted to 50 μl with PBS. The protein-inhibitor mix was incubated with 1 μg of Alu DNA. Simultaneously 3 μg of the anti-FLAG antibody (described above) was subjected to incubation with protein-G sepharose beads (60 μl, 17061801, GE Healthcare) for 1 hr with tumbling. The DNA-protein-inhibitor mix was transferred to the tube containing the anti-FLAG antibody-bound protein-G sepharose beads and incubated for 60 minutes at room temperature with tumbling. The beads were allowed to settle down followed by a gentle spin and the supernatant containing the unbound antibody and DNA was removed. The beads were gently washed three times with ice-cold 1x PBS. For each sample, the 1x TE buffer (40 μl per sample) was added and mixed followed by heating at 80°C for 20 minutes to elute the bound DNA.
The immunoprecipitated Alu DNA in presence and absence of Givinostat was used as a template for the qPCR (1725124, Biorad) using T7 and SP6 primers. The PCR was performed for both the samples (Alu DNA immunoprecipitated with Givinostat-inhibited and mock-inhibited rCGGBP1). The template was used at different dilutions of the immunoprecipitated DNA (1:100 and 1:200 diluted) for qPCR in multiple replicates. The input Alu DNA template was used as a control to calculate the first delta Ct (dCt). The second delta Ct values were calculated by subtracting the dCt values obtained for the mock-inhibited sample from those of the Givinostat-inhibited sample. Following are the PCR conditions for Alu PCR: 95°C-5 minutes, (95°C- 20 seconds, 55°C- 20 seconds, 72°C-30 seconds, 80°C-30 seconds) x50, melting curve analysis (50°C to 95°C with constant signal recording).
ChIP-qPCR
CGGBP1 ChIP was performed as described earlier [41]. HEK293T cells were treated with 2 μM Givinostat or DMSO for 6 hrs and 24 hrs. Approximately 1 million cells were crosslinked (1% formaldehyde solution, 37°C for 10 min, quenched with 125 mM Glycine) and harvested. After washing twice with PBS, the cell pellet was lysed in SDS lysis buffer supplemented with protease inhibitor cocktail (PI78441, Invitrogen). Resuspended and cleared chromatin was sonicated using a Diagenode Bioruptor (20 cycles of 30 seconds “ON”/30 seconds “OFF”). As per our previous experience, the sonication conditions were optimised for generating DNA fragments of length range 0.5 kb to 1 kb. The sonicated chromatin was centrifuged (16000 rcf, 5 minutes, 4°C) and 30 μl was set aside as input. CGGBP1 immunoprecipitation was performed on 120 μl of the cleared sonicated lysate diluted using ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) supplemented with protease inhibitor cocktail and precleared using protein G sepharose beads for 4 hrs at 4°C. The anti-CGGBP1 antibody was added and tumbled overnight at 4°C followed by protein G sepharose beads for 1hr. Protein G sepharose beads were collected by gentle centrifugation and washed with three buffers in the following order: low-salt IP wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl and 150 mM NaCl), high-salt IP wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl and 500 mM NaCl), LiCl IP wash buffer (0.25 M LiCl, 1% IGEPAL, 1% Na deoxycholate, 1 mM EDTA and 10 mM Tris–HCl). Finally, the Protein G sepharose beads were washed twice with TE buffer (10 mM Tris–HCl and 1 mM EDTA). The DNA bound to the Protein G sepharose beads was extracted in an elution buffer (1% SDS and 0.1 M NaHCO3), subjected to reverse crosslink (addition of 20 μl of 5 M NaCl and incubation at 65°C for 6 hrs), followed by addition of 10 μl of 0.5 M EDTA pH 8.0 and 20 μl of 1 M Tris-HCl pH 6.8, and then by the addition of 2 μl of 10 mg/ml Proteinase K (P2308, Sigma) and digestion for 1 hr at 42 °C. The ChIP-DNA was finally purified using the DNA purification kit (A1460, Promega) and used for qPCR. The purified ChIP-DNA was diluted 1:100 and used as a template for the qPCR. Following are the PCR conditions for Alu PCR: 95°C-5 minutes, (95°C- 20 seconds, 55°C- 20 seconds, 72°C-30 seconds, 80°C-30 seconds) x50, melting stage. The primers used for Alu PCR were 5’-GAGGCTGAGGCAGGAGAATCG-3’ and 5’-CGCCCAGGC TGGAGTGCAGTGGCGCG-3’. Similarly, CTCF and H3K4me3 ChIP-qPCRs were performed using primers for Alu as indicated above and primers for CTCF-binding sites (5’-CGTAGTTGGGCAGGTTGTCT-3’ and 5’-CAGCTAGGGGGCTACTTCCT-3’) covering the following genomic coordinates in hg38: chromosome 2:128372275-128372499. The PCR conditions were the same as those for the Alu except for the annealing temperature of 57°C.
Nucleo-cytoplasmic fractionation and Western blotting
The cytoplasmic and nuclear fractions from the HEK293T cells were separated by using the REAP protocol [49]. In brief, HEK293T cells were grown in 10 cm diameter cell culture dishes. Cells were treated with 2 µM Givinostat or DMSO for 24 hrs. Cells were harvested with a scraper and washed twice with ice-cold PBS. Cell pellets were resuspended in 250 µl ice-cold plasma membrane lysis buffer (PMLB) [PBS with 0.1% NP40 (PI78441, Sigma) and protease inhibitor cocktail (PI78441, Sigma)], mixed 3 times using a p1000 micropipette and centrifuged at 1200 rpm in a benchtop microfuge for 10 seconds. The supernatant was removed. The supernatant was centrifuged again at 1200 rpm 10 seconds and the clarified supernatant was used as the cytoplasmic fraction. The pellet was washed with PMLB and lysed in 250 µl ice-cold RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 8.0), 0.1% Sodium deoxycholate, 1% NP-40, 0.5% SDS) containing 1x Halt Protease Inhibitor cocktail (PI78441, Sigma) for 30 minutes. These nuclear lysates were cleared by centrifugation. Equal volumes of the cytoplasmic and nuclear fractions were subjected to SDS-PAGE followed by Western blotting. The levels of CGGBP1 in nuclear and cytoplasmic fraction were compared between mock (DMSO-treated) or Givinostat-treated HEK293T cells. GAPDH and Histone 3 antibodies (H3K4me3, H3K27me3, H3K9me3 and total Histone H3) were used to identify cytoplasmic and nuclear fractions respectively.
Histone extraction
HEK293T cells were grown in 10 cm cell culture dishes. Cells were harvested with a scraper and washed twice with ice-cold PBS. Cells were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v) and protease inhibitor cocktail (PI78441, Sigma)). Cells were lysed on ice for 10 minutes and centrifuged at 6,500 x g for 10 min at 4°C. The supernatant was discarded and the nuclei in the pellet were washed with TEB and centrifuged at 6,500 x g for 1 min at 4°C. The pellet was resuspended in 250 µl of 0.2 N HCl and left for acid extraction overnight at 4°C. Nuclear extracts were centrifuged at 6,500 x g for 10 min at 4°C and the clear supernatant was neutralised with 1/10 volume of the 2M NaOH. The pH of the neutralised extracts was adjusted to 7.5 by the addition of 1M Tris pH 6.8 and finally diluted 10 times with 1xPBS for DBID assays.
DNase activity determination
Genomic DNA was isolated from HEK293T cells. Genomic DNA (2 μg) was incubated with Givinostat (100 µM) or DNaseI (2 units) at room temperature for 5, 10 or 15 minutes. As a control, the same amount of genomic DNA was incubated for 15 minutes at room temperature without any Givinostat or DNaseI. The treatments (Givinostat or DNaseI) were quenched by adding 1 µl of 0.5 M EDTA to all samples. The degradation activity of the Givinostat was determined by agarose gel electrophoresis.