EXPERIMENT_TITLE: GSM7897983: K202Q Mutation Replicate 1 HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C libraries were prepared using in situ Hi-C protocol as previously described (Rao et al., 2014) with some modifications. In brief, 0.2 to 0.5 million HeLa cells were crosslinked with 16% formaldehyde (Thermo 28908) to a final concentration of 1% for 10 min at room temperature, followed by quenched by 0.2 M glycine for 5 min. Cell nuclei were extracted with lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% Igepal CA630) mixed with 50× protease inhibitor (Sigma P8340) on ice for at least 15 min. Cell pellets were resuspended in 0.5% SDS (prepared from 10% SDS  (Sigma 71736)) and incubated at 62°C for exactly 10 min, then quenched with 1.5% trix-100 (freshly prepared from 10% Triton X-100 (Sigma 648463)) for 15 min at 37°C. DNA was then digested with Dpn II restriction enzyme (NEB R0543L) for 16h at 37°C, followed by an incubation at 62°C for 20 minutes. End repair of DNA as well as biotin labeling was achieved by incubation at 37° for 1h in the fill-in master mix (with a final concentration of 0.4mM biotin-14-dATP (Invitrogen 19524016), 3.3 mM dCTP, 3.3 mM dGTP, 3.3 mM dTTP, 5000U/mL DNA Polymerase I, Large (Klenow) Fragment (NEB M0210L)). DNA was then ligated by incubation in a ligation master mix (composed of 664ul of water, 120μl of 10X T4 DNA ligase buffer (NEB B0202), 100μl of 10% Triton X-100 (Sigma 71736), 6μl of 20mg/ml Bovine Serum Albumin (Sigma B8667), and 10μl of 400 U/ μl T4 DNA Ligase (NEB M0202)) at room temperature with low-speed rotation for 4h. Cell pellets were resuspended in 300 μL of PK digestion buffer (10 mM Tris-HCl pH8.0, 0.5 M NaCl, 1% SDS), followed by the addition of 50 μL of 20mg/ml proteinase K (QIAGEN 19133). The mixture was digested at 55°C for 30 minutes to uncross-link the DNA, and then incubated overnight at 68°C. To efficiently purify the DNA, the TIANamp Genomic DNA Kit (TIANGEN DP304) was used instead of low-temperature precipitation with ethanol and sodium acetate. Sonication was then employed to break DNA into an average size of 300-500 bp. Biotin-labeled DNA was pulled down with Dynabeads MyOne Streptavidin C1 beads (Invitrogen 65001) and prepped for library construction. Libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme ND607) following the instructions.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: HDAC8, a member of class I HDACs, plays a pivotal role in cell cycle regulation by deacetylating the cohesin subunit SMC3. While cyclins and CDKs are well-established cell cycle regulators, our knowledge of other regulators remains limited. Here we reveal the acetylation of K202 in HDAC8 as a key cell cycle regulator responsive to stress. K202 acetylation in HDAC8, primarily catalyzed by Tip60, restricts HDAC8 activity, leading to increased SMC3 acetylation and cell cycle arrest. Furthermore, cells expressing the mutant form of HDAC8 mimicking K202 acetylation display significant alterations in gene expression, potentially linked to changes in 3D genome structure, including enhanced chromatid loop interactions. K202 acetylation impairs cell cycle progression by disrupting the expression of cell cycle-related genes and sister chromatid cohesion, resulting in G2/M phase arrest. These findings indicate the reversible acetylation of HDAC8 as a cell cycle regulator, expanding our understanding of stress-responsive cell cycle dynamics. Overall design: To directly examine the influence of K202 acetylation on cellular processes, we harnessed CRISPR/Cas9 technology to modify the HDAC8 gene in HeLa cells. We generated the K202Q missense mutation, which mimics the acetylated state, and concurrently, we also generated the K202R mutant. Monoclonal cells were screened and confirmed for successful mutation of the HDAC8 gene through DNA sequence analysis.
SAMPLE_TITLE: K202Q Mutation Replicate 1 HiC
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