Fig. 5: DNA-PKc-mediated phosphorylation of AF9 at Serine 395 (S395) residue reduces its interaction with SEC components for transcriptional downregulation during genotoxic stress within 293 T cells. | Nature Communications

Fig. 5: DNA-PKc-mediated phosphorylation of AF9 at Serine 395 (S395) residue reduces its interaction with SEC components for transcriptional downregulation during genotoxic stress within 293 T cells.

From: Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress

Fig. 5

A, B IR-induced dynamic phosphorylation of ectopically expressed AF9 ((A) n = 3 replicates) and endogenous AF9 ((B) n = 1 replicate). C, D Enhanced phosphorylation of ectopically expressed AF9 ((C), n = 3 replicates) and endogenous AF9 ((D) n = 2 replicates), and its concomitant reduced interaction with SEC components upon IR(10 Gy) treatment. E Interaction of ectopically expressed AF9 protein with endogenous DNA-PKc within mammalian cells (n = 3 replicates). F, G Effect of treatment of cells with NU7441(2 μM) on IR(10 Gy) treatment-dependent phosphorylation of ectopically expressed AF9 ((F) n = 2 replicates) and endogenous AF9 ((G) n = 3 replicates) and its concomitant interaction with SEC components G. H Immunoblotting analysis showing defective phosphorylation and unaltered SEC interaction in cells expressing phosphorylation-defective AF9 mutant (S395A) when compared to AF9(WT), upon IR(10 Gy) treatment (n = 3 replicates). I Effect of AF9(S395A) mutant on IR(10 Gy)-dependent phosphorylation and concomitant acetylation and its effect on interaction with SEC and TFIID components (n = 1 replicate). J Effect of AF9 phosphorylation mimic mutant (S395D) on its interaction with TFIID and SEC components (n = 1 replicate). K Nascent RNA transcription analysis showing enhanced global transcriptional activity in cells expressing AF9(S395A) mutant, when compared to AF9(WT) upon IR(10 Gy) treatment. The boxes represent median and quartiles and value ranges of 25–75 and 10–90%. Upper and lower hinges extend to the largest and smallest datapoints (n = 100 cells). This experiment was done once. L qRT-PCR analysis showing effect of re-expression of AF9(WT) and AF9(S395A) proteins in stable AF9 KD cells on mRNA expression of indicated AF9-target genes at 2hrs after IR(10 Gy) treatment (n = 2 replicates). M ChIP analysis showing recruitment of indicated factors at the promoter-proximal regions of AF9-target genes upon re-expression of AF9(S395A) in stable AF9 KD cells when compared to AF9(WT) at 2hrs after IR(10 Gy) treatment (n = 2 replicates). For this figure, the data showing RNA and ChIP analyses by qRT-PCR, the error bar represents mean ± SD and statistical analyses were performed using one-tailed Student’s t test. p values for each experimental data is mentioned on the bar diagram.

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