Genomic Profiling of Sirt6 binding sites in the neuroblastoma cell line ShSy-5y under normal culture conditions.
Histone deacetylases are important regulators of gene expression and chromatin conformation. Sirt6 is a histone deacetylase that regulates gene expression by deacetylating histone H3 on the specific lysine residues K9 and K56. Notably, Sirt6 expression and activity negatively correlate with biological aging and the onset of age-related diseases, such as Alzheimer's disease. Moreover, Sirt6 also binds to DNA double-strand breaks, as a DSB-sensor protein, and regulates the DNA damage response, promoting non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways. Therefore, Sirt6 functions as a stress signaling epigenetic regulator capable of coordinating the cellular response to several cytotoxic stimuli, including DNA damage, as one of its main regulatory outputs. We performed Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to investigate the Sirt6 genomic-binding profile in the neuroblastoma cell line ShSy-5y under normal culture conditions. This experiment allows the scientific community and us to identify the Sirt6 targets at the gene level, and predict a possible regulatory role on its expression. The study on Sirt6 binding to specific genes was further confirmed and analyzed by ChIP-qPCR under specific experimental conditions. Overall design: For the ChIPseq experiments, we did three independent biological replicates of the next experiment, including input samples and negative controls. We transduced Sh-Sy5y cells with a Myc-tagged Sirt6 protein. After transduction and puromycin selection, we performed the Chromatin Immunoprecipitations. The cells were plated for all the experimental conditions at an equal confluence, fixed with 1% formaldehyde for 10 minutes, quenched, and collected. After cell pellet resuspension in RIPA buffer, the lysates were sonicated using a vibracell needle sonicator (Sonics & Materials Inc, USA) to break the chromatin. Chromatin immunoprecipitations were carried out using anti-myc antibodies coated magnetic beads (MedChemExpress, Cat# HY-K0206) to isolate Sirt6-bound DNA. The negative controls consisted of untransduced ShSy-5y cells used for the same ChIP procedure, and the input samples were taken from the Sirt6-transduced samples used for Sirt6 ChIPs. The entire experiment was repeated on three independent occasions, and each one was validated by ChIP-qPCR using known Sirt6 target sequences. Immunoprecipitated DNA was then sequenced on a Novaseq 6000 sequencer (Illumina) using an SP 100 cycles kit, allocating 20M reads per sample, paired-end sequencing. Peaks were called using the MACS2 software, and the resulting peak files are included as processed data.