2.10. Chromatin Immunoprecipitation (ChIP) Assay

In this assay, proteins and interacting DNA are crosslinked with formaldehyde; the chromatin is sheared with either sonication mechanically or micrococcal nuclease enzymatically. The nucleoprotein complex is enriched by immunoprecipitation, and through the reversal of the crosslinking, DNA and the interacting protein are separated. In the end, the interacting DNA fragment is purified and quantified with ChIP-qPCR. To determine the promoter fragment to be amplified in ChIP PCR, Promo3.0 analysis used for predicting ets motifs and their dissimilarity scores was used (Supplementary Table S3). The amplicon size was arranged between 75–150 bp; CpG islands were checked for the potential binding sites of Elk-1, and the position of Elk-1 to those sequences was considered for primer design. The UCSC in silico PCR tool was used to verify the amplicon (https://genome.ucsc.edu/cgi-bin/hgPcr, accessed on 20 January 2021); primers used for ChIP PCR are listed in Table 5.

The list of primers used in chromatin immunoprecipitation (ChIP) assay.

Essentially, cells were seeded in three separate 150 mm cell culture dishes of 2 × 10⁶ cells/dish per experimental group on day zero. On day 1, cells were transfected with either an empty pCDNA3.1 plasmid or an expression plasmid for Elk1-VP16 plasmids and incubated 48 h at 37 °C, 5% CO₂. Cells were then treated with 1% formaldehyde at room temperature for 20 min; glycine was then added to the dishes to a final concentration of 0.125 M and incubated for 5 min at room temperature. The dishes were washed three times with cold PBS on ice and then centrifuged at 400× g for five minutes at 4 °C with 1× protease inhibitor cocktail (PIC) (Roche, 4693159001). The supernatant was aspirated, and lysis buffer was added onto the cells with a volume of at least 10 times the pellet obtained. The suspension was incubated on ice for 10 min and passed through an insulin needle 20 times. One volume of the sample was mixed with an equal volume of 0.4 percent Trypan Blue Dye, and the cell nuclei were checked under the microscope. The volume of the sonication buffer to be used to dissolve the pellet was adjusted to 2–3 × 10⁶ nuclei/mL and sonicated in the Biorupter UCD-200 Sonicator (Diagenode, Denville, NJ, USA). Following the sonication, cell lysates were centrifuged at 22,000× g for 20 min at 4 °C to remove insoluble materials. The supernatant was then diluted five-fold with dilution buffer and pre-cleared for 4 h with slow rotation with protein A/G mixture beads. After incubation, the samples were precipitated at 150× g for 5 min at 4 °C, and 10% of the total supernatant was removed as total input control and kept in −20 °C. The rest of the supernatant was divided into two fractions of the negative control (IgG-mock) and immunoprecipitation (IP) per group.

Sixty microliters of ANTI-FLAG^® M2 Affinity Gel (Sigma Aldrich, #A2220, Taufkirchen, Germany) resin per group were washed and equilibrated with five volumes of dilution buffer and centrifuged three times at 400× g for one minute each at 4 °C. The negative control and IP fractions separated from the dilution in the previous step were mixed with Protein G-Plus agarose beads and anti-Flag M2 resin, respectively. The tubes were incubated at 4 °C overnight with slow rotation. The following day, the mix was centrifuged at 4 °C and 600× g for five minutes, and the pellet was collected. The beads were washed with one mL of low salt, high salt, LiCl, and TE buffers at 4 °C with rotation, respectively. Following each of the washing steps, the beads were centrifuged at 4 °C and 600× g for five minutes.

At the elution step, the inputs that were collected and frozen a day before were thawed and added as the third fraction of each group. After the last wash, 250 μL fresh elution buffer, pre-heated at 65 °C, was added onto the beads, and they were incubated on a shaker for 15 min. The tubes were vortexed with five-minute intervals and then centrifuged at 4 °C and 18,000× g for five minutes. The supernatant was collected for each fraction of each group, and the elution step was repeated with another 250 μL elution buffer. After elution of the crosslinked DNA–protein complex, 10 μL of RnaseA (10 mg/mL) (Intron, #BR003) and 25 μL of 5 M NaCl was added onto the elutes and incubated for at least five hours or overnight at 65 °C. The following day, 10 μL of 0.5 M EDTA, 20 μL 1 M Tris–HCl (pH 6.5), and two μL Proteinase K (20 mg/mL) (Invitrogen, #25530049, Carlsbad, CA, USA) mix were added and incubated again at 65 °C for two more hours. Using MEGAquick-spin™ Plus Total Fragment DNA Purification Kit (Intron Bio, #17290, Sungnam, Korea), the DNA was cleaned up. The resulting fractions were used for qPCR analysis.

SSOAdvanced Universal SYBR Green Supermix (Bio-Rad, #1725274, Hercules, CA, USA) and Applied Biosciences StepOne Plus Real-Time System were used for qPCR analysis with DNA isolated from ChIP. Ten microliters of PCR reaction were prepared by mixing 2X SSO Advanced Universal SYBR Green Supermix, 300 nM forward and reverse primers each, and 1 µL template. In the analysis phase, qPCR signals obtained from the ChIP samples were normalized by the signals obtained from the input, and the mock samples and the results are presented as fold change. For statistical analysis, one-way ANOVA with Tukey post hoc test or Student’s t-test depending on the context with Prism 5 GraphPad software was used. p value under 0.05 was considered significant.