Journal: The Journal of Biological Chemistry

Article Title: p53-mediated regulation of LINE1 retrotransposon-derived R-loops

doi: 10.1016/j.jbc.2025.108200

Figure Lengend Snippet: Histone modification dynamics in L1 5′UTR-eGFP reporter cells upon HDACi exposure and withdrawal. A – D , chromatin immunoprecipitation followed by qPCR analyses was conducted on the 5′UTR region of A375 WT and p53 −/− L1 5′UTR-eGFP stable cells. Primers targeting the p53-binding region were utilized, with immunoprecipitation carried out using antibodies against histone modifications H3K9me3, H3K27me3, H3K9ac, and H3K4me3. The results illustrate levels of H3K9me3 ( A ) and H3K27me3 ( B ), representative of repressive histone marks, normalized to input values, as well as levels of activation marks H3K9ac ( C ) and H3K4me3 ( D ). Reporter cells were treated with HDACi (NaB drug) for 24 h, followed by withdrawal for 3 and 6 days. Samples for analyses were collected at first day, fourth day, and seventh day (for drug withdrawal), as depicted (refer to Fig. 5 ). It is noteworthy that alterations in H3K9me3, H3K27me3, H3K9ac, and H3K4me3 enrichment are specific to the p53 status (refer to Fig. S5 , A and B ). The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05, p ∗∗<0.01, and ns = non-significant. E , qPCR was performed to assess the knockdown of SETDB1 and G9A from the siRNA transfected WT and p53 −/− A375 L1 5′UTR-eGFP cell lines. The dark gray bar plot refers to the untransfected cells and the light gray being nontargeting control (NTC). The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05, and ns = non-significant. F and G , Western blotting was performed to assess the reduction in the expression levels of SETDB1 and G9A in the siRNA transfected samples of WT and p53 −/− A375 L1 5′UTR-eGFP cells. Please refer ( Fig. S5 , C and D ) for their quantification using Fiji software. H , qPCR to measure the transcript levels of L1 ORF1 in SETDB1 and G9A knocked down A375 WT and p53 −/− cell lines. The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant. I , ChIP-qPCR to quantify the deposition of H3K9me3 marks at L1 5′UTR in SETDB1 knocked down cells of A375 WT and p53 −/− L1 5′UTR-eGFP cells. The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant. J , ChIP-qPCR to quantify the deposition of H3K9me3 marks at L1 5′UTR in G9A knocked down cells of A375 WT and p53 −/− L1 5′UTR-eGFP cells.The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant; LINE1/L1, long interspersed nuclear element 1; HDACi, histone deacetylase inhibitor; NaB, sodium butyrate; ChIP, chromatin immunoprecipitation; qPCR, quantitative polymerase chain reaction.

Article Snippet: The siRNA sequences utilized for SETDB1 ( ) and G9A ( ) as described by along with the nontargeting siRNA control sequence (GenScript) are mentioned in .

Techniques: Modification, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY, Knockdown, Transfection, Control, Western Blot, Expressing, Software, ChIP-qPCR, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction