Figure 3.
PHF6 protects from replication-associated DNA damage and binds to satellite DNA heterochromatin. (A) Schematic of chlorodeoxyuridine (CldU, red)/iododeoxyuridine (IdU, green) pulse labeling (upper left). Representative images of CldU and IdU replication tracks in Jurkat control (EV, control sgRNA) or PHF6 knockout (KO) cells (bottom left). Fork rate dot plot showing the IdU tract length of individual replication forks in untreated Jurkat cells (right). The median value of >350 tracts per experimental condition is indicated. Statistical analysis was conducted by using the Mann-Whitney test (P < .0001). Data are representative of 2 independent experiments. (B) Western blot showing PHF6 KO in Jurkat cells infected with a control sgRNA (EV) or a single guide RNA targeting the second PHD2 domain of PHF6 (KO). β-actin concentrations are shown as a loading control. (C) Left, scheme of the signals used for quantification of asymmetry analysis of forks moving from a single origin (outgoing forks). Right, scatter diagram of fork symmetry in Jurkat cells. Each dot corresponds to the ratio between the right and the left fork velocities of a pair of outgoing forks belonging to the same replication bubble. The areas outside of the dotted lines include all points whose ratios deviate from the expected theoretical value of 1 ± 0.3, corresponding to forks moving bidirectionally at nearly the same rate. EV control (control sgRNA), PHF6 KO (sgRNA targeting second PHD2 domain of PHF6). Statistical analysis was performed with the Mann-Whitney rank sum test (P < .001). (D) Western blot showing the presence of phosphorylated RPA (pRPA) after recovery from 30 minutes of 1 μM camptothecin treatment. The “–” sign indicates untreated conditions. Both RPA total amount and β-actin concentrations are shown as a loading control as used for the blot quantification shown below each band. EV control (control sgRNA), PHF6 KO (sgRNA targeting second PHD2 domain of PHF6). (E) Chromosome 19 distribution of normalized PHF6 (red track) or H3K9me3 (blue track) ChIP-Seq intensities in Jurkat. (F) Heat map indicating the log2FC enrichment in repetitive regions according to category compared with the average in 3 random subsets. (G) Overlap between PHF6 peaks and γ-H2AX genomic regions in untreated (upper panel) and after aphidicolin treatment (lower panel). The indicated P value and Z scores are the result of permutation testing (n = 1000 trials). PHF6 control obs, refers to observed regions. PHF6 control perm, refers to median expected permutated regions. (H) Normalized ChIP-Seq heat maps of Jurkat PHF6 control and KO and K562 γ-H2AX aphidicolin treated and untreated. PHF6-bound regions (n = 11 528) were scaled to the same length. (I) Differential PHF6 (control/KO cell lines) and γ-H2AX (treated/untreated) ChIP-Seq intensities within the fragile site FRA3H.

PHF6 protects from replication-associated DNA damage and binds to satellite DNA heterochromatin. (A) Schematic of chlorodeoxyuridine (CldU, red)/iododeoxyuridine (IdU, green) pulse labeling (upper left). Representative images of CldU and IdU replication tracks in Jurkat control (EV, control sgRNA) or PHF6 knockout (KO) cells (bottom left). Fork rate dot plot showing the IdU tract length of individual replication forks in untreated Jurkat cells (right). The median value of >350 tracts per experimental condition is indicated. Statistical analysis was conducted by using the Mann-Whitney test (P < .0001). Data are representative of 2 independent experiments. (B) Western blot showing PHF6 KO in Jurkat cells infected with a control sgRNA (EV) or a single guide RNA targeting the second PHD2 domain of PHF6 (KO). β-actin concentrations are shown as a loading control. (C) Left, scheme of the signals used for quantification of asymmetry analysis of forks moving from a single origin (outgoing forks). Right, scatter diagram of fork symmetry in Jurkat cells. Each dot corresponds to the ratio between the right and the left fork velocities of a pair of outgoing forks belonging to the same replication bubble. The areas outside of the dotted lines include all points whose ratios deviate from the expected theoretical value of 1 ± 0.3, corresponding to forks moving bidirectionally at nearly the same rate. EV control (control sgRNA), PHF6 KO (sgRNA targeting second PHD2 domain of PHF6). Statistical analysis was performed with the Mann-Whitney rank sum test (P < .001). (D) Western blot showing the presence of phosphorylated RPA (pRPA) after recovery from 30 minutes of 1 μM camptothecin treatment. The “–” sign indicates untreated conditions. Both RPA total amount and β-actin concentrations are shown as a loading control as used for the blot quantification shown below each band. EV control (control sgRNA), PHF6 KO (sgRNA targeting second PHD2 domain of PHF6). (E) Chromosome 19 distribution of normalized PHF6 (red track) or H3K9me3 (blue track) ChIP-Seq intensities in Jurkat. (F) Heat map indicating the log2FC enrichment in repetitive regions according to category compared with the average in 3 random subsets. (G) Overlap between PHF6 peaks and γ-H2AX genomic regions in untreated (upper panel) and after aphidicolin treatment (lower panel). The indicated P value and Z scores are the result of permutation testing (n = 1000 trials). PHF6 control obs, refers to observed regions. PHF6 control perm, refers to median expected permutated regions. (H) Normalized ChIP-Seq heat maps of Jurkat PHF6 control and KO and K562 γ-H2AX aphidicolin treated and untreated. PHF6-bound regions (n = 11 528) were scaled to the same length. (I) Differential PHF6 (control/KO cell lines) and γ-H2AX (treated/untreated) ChIP-Seq intensities within the fragile site FRA3H.

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