Figure 6A Newly synthesized proteins pulse - labeled with 35S - methionine for 10 min in HeLa cells pre - treated with indicated compounds for 2 . 5h , except ( E ) ( Bottom ) Coomassie - stained gel . B Newly synthesized proteins in HeLa cells treated with 2 . 5 μg / ml Tm or 50 μg / ml cycloheximide ( CHX ) . C Newly synthesized proteins in 4KO or 4KO + GCN2 MEF cells treated with 12 . 5 or 200 nM HF . D Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with 12 . 5 or 200 nM HF . E Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with indicated concentrations of Tm for 2 . 5 or 5 h . F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation ( 10 mM ) . H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) . I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom) Coomassie-stained gel.B Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX).C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF.D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF.E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).


Figure 7G , H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 ( G ) and H23 ( H ) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose - response ( IC50 ) . Each point represents mean ( n = 3 biological replicates ) ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7G, H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 (G) and H23 (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.


Figure 1 ( D ) U2OS ( EGFP - BIR - 5085 ) cells expressing shRNAs for RAD51 , POLD3 or shRNA vector ( Ctrl ) were infected by lentiviruses encoding endonuclease I - SceI . The percentage of EGFP positive cells was assayed by FACS analysis 4 days later ( left ) . Expression of RAD51 and POLD3 is shown by Western blot analysis ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * * P < 0 . 0001 and n . s . ( not significant ) P > 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) U2OS (EGFP-BIR-5085) cells expressing shRNAs for RAD51, POLD3 or shRNA vector (Ctrl) were infected by lentiviruses encoding endonuclease I-SceI. The percentage of EGFP positive cells was assayed by FACS analysis 4 days later (left). Expression of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ****P < 0.0001 and n.s. (not significant) P>0.05.


Figure 2 ( B ) U2OS WT or PIF1 - KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min . Labeled cells were processed for DNA fiber analysis . Representative images of stalled or restarted forks and forks with new origin firing were shown ( left ) . The percentage of restarted forks was quantified by analyzing of 110 - 130 fibers for each experiment ( right ) . Experiments were repeated four times for each sample . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 . ( D ) U2OS ( EGFP - BIR - 5085 ) cell lines carrying Dox - inducible Cas9 / sgRNA - 5085 ( Dox - Cas9 ) or Cas9n / sgRNA - 5085 ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) and assayed by FACS analysis 2 days later ( left ) . U2OS ( EGFP - BIR - 5085 , Dox - Cas9 or Dox - Cas9n ) cells expressing shRNAs RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were incubated with 5 µg / ml Dox , and FACS analysis was performed after 2 days ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.(D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.


Figure 3 ( E ) U2OS ( EGFP - Flex1 - BIR - 5086 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were infected by retroviruses expressing RAS , and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection ( top ) . Expression level of RAD51 and POLD3 is shown by Western blot analysis ( bottom ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 . ( F ) U2OS WT or PIF1 - KO cells were infected by retrovirus expressing RAS or empty vector . Cell profiling was plotted by counting cell numbers every 24 hr , and normalized to the cell number on the first day . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(E) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.(F) U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 hr, and normalized to the cell number on the first day. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


Figure 4 ( C ) U2OS ( EGFP - STGC - 1731 ) cell lines carrying Dox - inducible Cas9 / sgRNA - 1731 ( Dox - Cas9 ) or Cas9n / sgRNA ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later ( top ) . U2OS ( EGFP - STGC - 1731 , Dox - Cas9 or Dox - Cas9n ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA control ( Ctrl ) were incubated with Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days ( bottom ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 . ( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were treated with or without 2 mM HU for 24 hr , and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were treated with 2 mM HU for 24 hr , and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 . ( F ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were infected by retroviruses expressing RAS or empty vector , and the percentage of EGFP positive cells was assayed by FACS 5 days following infection ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS , and the percentage of EGFP positive cells was assayed by FACS 5 days after infection ( middle ) . Expression level of RAD51 and POLD3 is shown by Western blot analysis ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


Figure 5 ( D ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 . ( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were treated with 2 mM HU for 24 hr . The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal ( left ) . U2OS ( EGFP - STGC - 1731 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were infected by lentivirus expressing I - SceI . The percentage of EGFP positive cells by I - SceI induction was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 . ( F ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 . ( G ) FLAG - PIF1 was stably expressed in U2OS ( EGFP - Flex1 - STGC - 1541 ) cells by lentiviral infection . Enrichment of FLAG - PIF1 at Flex1 site or GAPDH site with or without HU ( 2 mM , 10 hr ) treatment was calculated by anti - FLAG ChIP ( left ) . When PCNA was depleted by shRNA using vector as a control ( Ctrl ) , enrichment of FLAG - PIF1 at Flex1 site was calculated by anti - FLAG ChIP ( right ) . ChIP value in cells without HU treatment is set up as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(D) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(F) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


Figure 6 ( D ) Growth curves of WT or FANCM - KO HCT116 cells were plotted after expressing POLD3 ( left ) or PIF1 ( right ) shRNA or shRNA vector ( Ctrl ) . Cell number was normalized to that on day one . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


Figure 7 ( C ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 . ( E ) U2OS ( EGFP - BIR - 5085 ) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I - SceI . The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I - SceI infection . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 . ( I ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells , or PIF1 - KO cells expressing PIF1 - WT , and E307Q and L319P mutants before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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Figure 7(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


Figure 1Figure 1 . Identification of non - canonical p53 binding sites in human subtelomeres . ( A and B ) ChIP - seq tracks for p53 in camptothecin ( CPT ) treated or untreated HCT116 cells were aligned with RNAPII , CTCF , RAD21 , and SMC1 for human chromosome18q ( A ) or 13q ( B ) subtelomeres . Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top . Subtelomeric DNA paralogy segment tracks , internal telomere - like sequence tracks ( red CCCTAA ) , and annotated RefSeq gene tracks are indicated above the ChIP - seq tracks . ( C ) Alignment of human subtelomeric elements containing non - canonical p53 recognition sequences . The position of the RepeatMasker - defined LTR10c element is shown ; also note the ( TG ) n microsatellite repeat adjacent to the p53 binding site . ( D ) Schematic organization of non - canonical p53 binding sites found in human subtelomeres .

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Figure 1Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.


Figure 2Stress - induced p53 binding at human subtelomeres . ( A ) Western blot of HCT116 p53 - / - and p53 + / + cells treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hrs . Cell lysates were assayed for total p53 , phospho - Ser15 p53 ( p - p53 ) , γH2AX , or GAPDH . ( B ) ChIP - qPCR was assayed in HCT116 p53 - / - or p53 + / + cells treated with DMSO or etoposide as in panel A , and assayed by ChIP with antibodies to p53 ( Ab6 ) or control IgG , followed by qPCR for p53 binding site in subtelomere 18q ( top panel ) or 13q ( lower panel ) . ( C ) HCT116 p53 - / - and p53 + / + cells were cultured in DMEM with 10 % FBS or Hank ' s buffered saline solution ( HBSS ) lacking FBS and then assayed by ChIP with either IgG control ( grey ) or two different p53 - specific antibodies Ab6 ( red ) or FL ( black ) . ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q , 13q , or p21 promoter . . ( D ) Doxycyclin ( Dox ) - inducible p53 H1299 cells were treated without ( - ) or with ( + ) Dox in media containing 10 % serum or in serum free HBSS , and then subject to ChIP assay with control IgG or p53 - specific Ab6 antibody . ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 ( top panel ) , p53 - negative control IGX1A ( middle panel ) , or p21 positive control ( lower panel ) . Error bars represent SD , and * indicates p value < . 05 . p - values were determined by Student t - test .

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Figure 2Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH.(B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel).(C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter.. (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.


Figure 3 . Transcriptional activity of subtelomeric p53 - response elements . ( A ) Luciferase test constructs containing either empty test vector ( top ) , p53 - response elements from 18q subtelomere ( middle ) or MDM2 promoter ( lower ) were assayed in HCT p53 - / - cells transfected with two independent preparations of empty vector or p53 WT expression vector . ( B ) RT - qPCR measure of p21 ( top panel ) or PARD6G ( lower panel ) in HCT116 p53 - / - or p53 + / + cells after treatment with DMSO or 50 µM etoposide . ( C ) RT - qPCR analysis of TERRA transcripts from subtelomere 13q ( upper ) or 2q ( lower ) . ( D ) RT - qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 ( top panel ) or pr2 ( lower panel ) from HCT116 p53 + / + cells cultured with serum ( grey bars ) or in HBSS lacking serum ( black bars ) . Error bars represent SD and * indicates p value < . 05 using student t - test comparing treated and untreated samples .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector.(B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide.(C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower).(D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.


Figure 4 . p53 - dependent cell viability and telomere protection in response to DNA damage . ( A ) Western blot of total p53 , γH2AX , and GAPDH in HCT116 p53 - / - or p53 + / + cells treated with 1 µM or 50 µM etoposide for 24 hrs . s . ( B ) Cell cycle analysis of HCT116 p53 - / - or p53 + / + cells untreated or treated with 50 µM etoposide for 24 hrs . Cell cycle was determined by FACS analysis of propidium iodide stained cells . ( C ) Cell clonal survival assay for HCT116 p53 - / - or p53 + / + cells treated with DMSO or 1 , 1 . 5 or 2 µM etoposide for 24 hr prior to replating . . ( D ) Quantification of clonal survival assay relative total cell numbers ( top panel ) or normalized to DMSO controls ( lower panel ) . E ) TIF assay for HCT116 p53 + / + or p53 - / - cells treated with 1 µM etoposide for 3 hrs . Telomere DNA FISH with TelC probe ( red ) , γH2AX ( green ) , Dapi ( blue ) . ( F ) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53 + / + ( black ) or p53 - / - ( red ) cell . G ) Telomere length assay showing p53 - / - or p53 + / + cells treated with either DMS0 , 1 or 50 µM etoposide . Ethidium bromide ( EtBr ) stained agarose gels ( left panel ) , Southern blot hybridized with Tel C probe ( middle panel ) or with α Satellite DNA promote ( right panel ) . Quantification of telomereDNA signal relative to invariant bands in α Sat . Error bars represent SD and * indicates p values < . 05 determined by student t - test ( TLA ) , or chi - square ( TIF assay ) . ( H ) Two - dimensional agarose gel analysis of telomeric DNA from HCT p53 + / + or or p53 - / - treated with 50 µM etoposide for 24 hrs . Ethidium gel ( top ) and Southern blot probed with TelG probe ( lower panel ) . Single stranded telomere DNA ( red arrow ) and recombination / replication - associated structures ( blue arrow ) are indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs.s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells.(C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating.. (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel).E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue).(F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell.G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay).(H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.


Figure 5Figure 5 . p53 - dependent histone modification changes in response to DNA damage stress . HCT116p53 - / - ( left panels ) or p53 + / + ( right panels ) were treated with DMSO ( black ) or 50 µM etoposide ( red ) for 24 hrs and assayed by ChIP - qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere ( panel A ) or 13qsubtelomere ( panel B ) or for control regions ( p21 promoter , GKN1 / 2 , PARKIN ) as indicated below . ChIP - for p53 , H3K9Ac , H3K27Ac , γH2AX , or IgG control were quantified as % input . Error bars indicates SD and * indicates p value < . 05 using student t - test comparing ChIP values in p53 + / + relative to p53 - / - cells for specific primer pairs . ( C ) Dot blot analysis of telomere repeat or Alu repeat DNA from p53 + / + or p53 - / - HCT116 cells treated with DMSO or etoposide ( ETP ) ( as described in panel A ) , that were subject to ChIP with antibodies to IgG , p53 , γH2AX , TRF2 , or TRF1 . Quantitation of three independent ChIP - assays for TRF1 and TRF2 is shown in the panel to the right . ( ( E ) ChIP - qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q ( left ) or 13q ( right ) .

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Figure 5Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP- for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs.(C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. ((E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).


Figure 6 ( A ) HCT116 p53 + / + or p53 - / - cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53 , γH2AX , H3K9Ac , panH3 , and GAPDH at times 0 , 8 , 16 , or 24 hr post - treatment . ( B ) ChIP - qPCR for γH2AX ( upper panels ) or IgG control ( lower panels ) for cells treated as indicated in panel A . ( C - D ) RT - qPCR analysis for cells treated as shown in panel A . C - D ) RT - qPCR analysis for cells treated as shown in panel A . ( C ) RT - PCR for the p21 ( top ) , or PARD6G mRNA ( lower ) . ( D ) RT - PCR for primer pairs spanning subtelomere18q as indicated below . Lower panel shows the No - RT control for 18qsubtelomere primers . RT - qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value < . 05 using student t - test comparing etoposide ( 24 hr ) relative to untreated for specific primer pairs . ( E ) Meta - data analysis of published GRO - Seq and ChIP Seq data sets . Gro - Seq Minus ( red / maroon ) or Plus ( purple / pink ) RNA strand from Control or Nutlin - 3a treated MCF7 cells was mapped to human 18q subtelomere . Histone H3K27ac , H3K4me1 , and H3K4me3ChIP - Seq data sets from untreated MCF7 are also shown for these subtelomeric regions . Right panel shows a zoom in of approximately 1000bp ( light blue on left panel ) surrounding the p53 response element within the subtelomere . ( F ) Summary of GRO - Seq and ChIP - Seq metadata analyses from human subtelomeres with p53 binding sites . Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself .

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Figure 6(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment.(B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A.C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs.(E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere.(F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.


Figure 7 ( B ) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hr . Blots were probed with pan - p53 , phospho - p53 , γH2AX , or GAPDH ( as loading control ) ( C - E ) RT - qPCR for p21 ( C ) , PARDG6 ( D ) , or 18q subtelomeric RNA ( E ) in 18q WT or18q Δp53 treated with DMSO ( black ) or 50 μM etoposide ( red ) for 24 hr . ( F ) ChIP - qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B - E .

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Figure 7(B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 µM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control)(C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.


Figure 1 ( A ) Fractionation of nuclear and cytosolic compartments using a standard NP - 40 lysis protocol and immunoblotting with chaperone specific antibodies ( B ) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in ( A ) . Cells are segregated into those undergoing replication and those that are not . Scale bar represents 5 μm ( D ) Real - time imaging of cells undergoing biochemical fractionation . sNASP is tagged with mCherry , whilst LamnA / C is tagged with EGFP . The left column of panels represents a maximum intensity projection . The time course on the right represents a plane in the z dimension reconstructed from 20 z - stacks penetrating 20 μm into the medium . Scale bars represent 5

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 (A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies  (B) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in (A). Cells are segregated into those undergoing replication and those that are not. Scale bar represents 5 μm (D) Real-time imaging of cells undergoing biochemical fractionation. sNASP is tagged with mCherry, whilst LamnA/C is tagged with EGFP. The left column of panels represents a maximum intensity projection. The time course on the right represents a plane in the z dimension reconstructed from 20 z-stacks penetrating 20 μm into the medium. Scale bars represent 5 μm


Figure 6 ( A ) HeLa cell lines stably expressing eGFP - sNASP , eGFP - ASF1 and eGFP - HAT1 . Scale bars represent 10 μm ( B ) Histone chaperone immunoprecipitations separated by 15 % SDS - PAGE and stained with Coomassie . Major bands that could be confidently identified by mass spectrometry are indicated . Due to the high percentage of acrylamide used , many of the higher molecular weight bands are not resolved . Asterisks represent partial degradation . Background - corrected densitometry profiles are shown for the portions of the gels covering H3 and H4 . Gel filtration purified recombinant H3 - H4 dimer is shown on the

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 (A) HeLa cell lines stably expressing eGFP-sNASP, eGFP-ASF1 and eGFP-HAT1. Scale bars represent 10 μm (B) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right


Figure 1 ( A ) MOG1 interacts genetically with the H2B deubiquitination machinery . Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures ( B ) Expression levels of total H2B and H2Bub1 were analyzed in wild - type ( WT ) , mog1Δ , ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti - total H2B and anti - H2Bub1 antibodies . Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments . The P - value was calculated using Student ´ s t - test . Error bars represent SD . The P - value was calculated using Student ´ s t - test ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 ) Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures ( C ) , ( D and were then spotted on YPD or YPGal and incubated for 2 daysTen - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures ( E and were then spotted on YP and incubated for 2

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) MOG1 interacts genetically with the H2B deubiquitination machinery. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures(B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (C), (D and were then spotted on YPD or YPGal and incubated for 2 daysTen-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (E and were then spotted on YP and incubated for 2 days


Figure 2 ( A ) The expression levels of H3K4me3 and H3 in total extracts of wild - type ( WT ) , mog1Δ , set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies . Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments . The P - value was calculated using Student ´ s t - test ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 ) ( B ) ChIP analysis of H3K4me3 presence at ADH1 , PMA1 and YEF3 promoter ( P ) and 5 ' ORF in wild - type ( WT ) or mog1Δ strains relative to total H3 . The bar graphs indicate the mean and standard deviation for at least three independent experiments . The significance of the differences was calculated using Student ´ s t - test ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 ) ( C ) MOG1 interacts genetically with the H3 methylases Set1 and Set2 . Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures . ( D ) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in ( C )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) The expression levels of H3K4me3 and H3 in total extracts of wild-type (WT), mog1Δ, set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies. Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)(B) ChIP analysis of H3K4me3 presence at ADH1, PMA1 and YEF3 promoter (P) and 5'ORF in wild-type (WT) or mog1Δ strains relative to total H3. The bar graphs indicate the mean and standard deviation for at least three independent experiments. The significance of the differences was calculated using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P < 0.001)(C) MOG1 interacts genetically with the H3 methylases Set1 and Set2. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures. (D) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in (C)


Figure 3 ( A ) Ten - fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea ( HU ) and were incubated for 2 - 3 days . ( B ) mRNA synthesis rates ( SR ) as determined in Genomic Run - On analysis and ( C ) mRNA abundance ( RA ) , in mog1 ∆ compared with wild type ( WT ) cells . Bar graphs showing the average level and standard deviation of three experiments ( n = 3 ) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units ( x 107 ) from image analysis quantification . Error bars represent SD . Significance of the differences was obtained using Student ´ s t - test and statistical difference is considered as * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P value < 0 . 001 . The scatter plots ( right panels ) show the variation of the transcription ( B ) or mRNA ( C ) levels of individual genes . Pearson correlation values are : - 0 . 039 ( SR ) and - 0 . 042 ( RA ) . This low correlation and the flat shape of the clouds on scatter plots ( see red tendency line ) indicates that there is no bias of the transcriptional effect with regard to expression level . ( D ) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes ( Total ) , SAGA and TFIID - dominated genes . These data were obtained from three independent replicates ( n = 3 ) of the RA and SR data that were averaged as explained in the main text . The median and standard deviation for the whole data set ( Total ) and for 351 ( for RA ) or 316 ( SR ) SAGA - dominated genes and for the 3903 ( RA ) or 3403 ( SR ) TFIID - dominated genes are shown . Significance of the differences was calculated using Student ´ s t - test . No significant difference between the different groups was found . The similar variations in SRs and RAs indicate that there are no changes in mRNA half - lives upon MOG1 deletion ( shown in Fig EV4 ) . The total number of genes with confident value in the analyses was 3953 for RA and 3989 for

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Ten-fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea (HU) and were incubated for 2-3 days.(B) mRNA synthesis rates (SR) as determined in Genomic Run-On analysis and (C) mRNA abundance (RA), in mog1∆ compared with wild type (WT) cells. Bar graphs showing the average level and standard deviation of three experiments (n=3) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units (x 107) from image analysis quantification. Error bars represent SD. Significance of the differences was obtained using Student´s t-test and statistical difference is considered as *, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P value < 0.001. The scatter plots (right panels) show the variation of the transcription (B) or mRNA (C) levels of individual genes. Pearson correlation values are: -0.039 (SR) and -0.042 (RA). This low correlation and the flat shape of the clouds on scatter plots (see red tendency line) indicates that there is no bias of the transcriptional effect with regard to expression level. (D) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes (Total), SAGA and TFIID-dominated genes. These data were obtained from three independent replicates (n=3) of the RA and SR data that were averaged as explained in the main text. The median and standard deviation for the whole data set (Total) and for 351 (for RA) or 316 (SR) SAGA-dominated genes and for the 3903 (RA) or 3403 (SR) TFIID-dominated genes are shown. Significance of the differences was calculated using Student´s t-test. No significant difference between the different groups was found. The similar variations in SRs and RAs indicate that there are no changes in mRNA half-lives upon MOG1 deletion (shown in Fig EV4). The total number of genes with confident value in the analyses was 3953 for RA and 3989 for SR


Figure 4 ( A ) ChIP analysis of TAP - tagged Rad6 presence at ADH1 , PMA1 and YEF3 coding regions ( 5 ' & 3 ' ) in WT or mog1Δ strains . Bar charts indicate the mean and standard deviation for at least three independent experiments . Significance of the differences was obtained using Student ´ s t - test ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 , * * * , P value = 0 . 0001 - 0 . 001 , * * * * , P value < 0 . 0001 ) . ( B ) , ( C ) and ( D ) Bre1 - TAP , Rtf1 - TAP and Rpb3 respectively , were analyzed as described in ( A ) . ( E ) Bar charts showing the relative chromatin occupancy to 5 ' ORFs ( left panel ) or 3 ' ORFs ( right panel ) of each factor relative to Rpb3 occupancy at these gene positions .

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Figure 4(A) ChIP analysis of TAP-tagged Rad6 presence at ADH1, PMA1 and YEF3 coding regions (5' & 3') in WT or mog1Δ strains. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01, ***, P value = 0.0001-0.001, ****, P value <0.0001). (B), (C) and (D) Bre1-TAP, Rtf1-TAP and Rpb3 respectively, were analyzed as described in (A). (E) Bar charts showing the relative chromatin occupancy to 5'ORFs (left panel) or 3'ORFs (right panel) of each factor relative to Rpb3 occupancy at these gene positions.


Figure 5 ( A ) Co - purification of Rtf1 - PK in cells expressing Mog1 - TAP ( + , + ) . A non - tagged strain ( - , - ) and cells expressing only Rtf1 - PK ( + , - ) were included as negative controls for co - purification . Inputs and IPs are depicted for each strain . ( B ) ChIP analysis of the presence of HA - tagged Mog1 compared to a non - tagged strain ( WT ) at PMA1 and YEF3 promoter ( P ) and coding regions ( 5 ' ORF ) . The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region . Bar charts indicate the mean and standard deviation for at least three independent experiments . Significance of the differences was obtained using Student ´ s t - test and is presented as a P - value ( * , P < 0 . 05 ) . ( D ) Mog1 - HA immunoprecipitation in cells expressing Bre1 - PK ( upper panel ) and Mog1 - TAP co - precipitation in cells expressing Ada2 - HA ( lower panel ) are indicate as in ( A ) Inputs and IPs are depicted for each strain .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Co-purification of Rtf1-PK in cells expressing Mog1-TAP (+,+). A non-tagged strain (-,-) and cells expressing only Rtf1-PK (+,-) were included as negative controls for co-purification. Inputs and IPs are depicted for each strain.(B) ChIP analysis of the presence of HA-tagged Mog1 compared to a non-tagged strain (WT) at PMA1 and YEF3 promoter (P) and coding regions (5'ORF). The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test and is presented as a P-value (*, P<0.05).(D) Mog1-HA immunoprecipitation in cells expressing Bre1-PK (upper panel) and Mog1-TAP co-precipitation in cells expressing Ada2-HA (lower panel) are indicate as in (A) Inputs and IPs are depicted for each strain.


Figure 6Representative images of the analysis of nuclear mRNA export are shown . The cellular localization of poly ( A ) + RNA was assessed by in situ hybridization using Cy3 - labeled oligo ( dT ) probes ( red ) in the indicated strains . In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours . DNA was stained by DAPI ( blue ) . In all images the scale bar corresponds to 2 μM . Images are representative pictures from 2 independent experiments of approximately 600 cells . The percentages indicate the number of cells showing mRNA export defect from these experiments .

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Figure 6Representative images of the analysis of nuclear mRNA export are shown. The cellular localization of poly(A)+RNA was assessed by in situ hybridization using Cy3-labeled oligo(dT) probes (red) in the indicated strains. In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours. DNA was stained by DAPI (blue). In all images the scale bar corresponds to 2 μM. Images are representative pictures from 2 independent experiments of approximately 600 cells. The percentages indicate the number of cells showing mRNA export defect from these experiments.


Figure 7 ( A ) The Mog1E65K Ran - binding mutant complements the slow growth of mog1ΔsetΔ mutants . Ten - fold serial dilutions of wild - type ( WT ) , mog1Δ , set1Δ , and mog1ΔsetΔ strains transformed with wild - type MOG1 ( pMOG1 ) or with the Ran - binding mutant ( pMOG1E65K ) as indicated were spotted on SC - LEU and incubated for 2 days at the indicated temperatures . ( B ) The Mog1E65K Ran - binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ . The expression levels of H3K4me3 , total H3 , H2Bub1 , total H2B and PGK1 in whole cell lysates from wild type ( WT ) or from mog1Δ cells transformed with an empty plasmid ( + pEmpty ) , wild type MOG1 ( pMOG1 ) or a Ran - binding mutant ( pMOG1E65K ) were analyzed by western blotting using the corresponding antibodies . ( C ) Gsp2 is not involved in H2Bub1 regulation . The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type ( WT ) and gsp2Δ strains by western blotting using anti - total H2B and anti - H2Bub1 antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) The Mog1E65K Ran-binding mutant complements the slow growth of mog1ΔsetΔ mutants. Ten-fold serial dilutions of wild-type (WT), mog1Δ, set1Δ, and mog1ΔsetΔ strains transformed with wild-type MOG1 (pMOG1) or with the Ran-binding mutant (pMOG1E65K) as indicated were spotted on SC-LEU and incubated for 2 days at the indicated temperatures.(B) The Mog1E65K Ran-binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ. The expression levels of H3K4me3, total H3, H2Bub1, total H2B and PGK1 in whole cell lysates from wild type (WT) or from mog1Δ cells transformed with an empty plasmid (+pEmpty), wild type MOG1 (pMOG1) or a Ran-binding mutant (pMOG1E65K) were analyzed by western blotting using the corresponding antibodies.(C) Gsp2 is not involved in H2Bub1 regulation. The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type (WT) and gsp2Δ strains by western blotting using anti-total H2B and anti-H2Bub1 antibodies.


Figure 1B . and C . Molecular models based on the crystal structures previously published [ 44 ] represent configurations of two dimers on DNA : Sox2 - Oct4 heterodimer ( B ) and Oct6 - Oct6 homodimer ( C ) . Oct4 POU domain , green ; Oct4 / Oct6 linker region , magenta ; Sox2 HMG , cyan ; Oct6 POU domain , orange . Individual helixes of POU domains are numbered . In both models , the DNA is represented as a light grey surface . Unless indicated otherwise , the representation is kept throughout the manuscript . D . EMSAs with a 2 - fold dilutions series starting at 2 μM of the Oct4 POU ( left ) or the Oct6 POU ( right ) in the presence of cy5 - labeled MORE DNA . The free DNA and DNA bound as monomer or dimer are indicated . E . and F . Bar plots of EMSA - derived cooperativity factors for Oct - Oct homodimers on the MORE ( E ) or Sox2 - Oct heterodimers on the SoxOct element ( F ) are shown for a panel of six POU proteins . The mean is shown with standard deviation as error bars ( n ≥ 3 ) and Tukey multiple comparison of means was used for assessment of statistical significances ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) . N . A . : not assessed . G . Properties of Oct4 and Oct6 homodimers from molecular dynamics ( MD ) simulations . The structural snapshots illustrate Oct6 ( first monomer in orange , second in dark blue ) and the 3 parameters used : drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains ( excluding the linker ) ; θ = the angle describing the kink in helix α4 ( data for both monomers 1 and 2 are shown ) ; d = the distance between the centers of mass of the 2 helices α5 ; w = the DNA minor groove width in the region between the 2 monomers .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. and C. Molecular models based on the crystal structures previously published [44] represent configurations of two dimers on DNA: Sox2-Oct4 heterodimer (B) and Oct6-Oct6 homodimer (C). Oct4 POU domain, green; Oct4/Oct6 linker region, magenta; Sox2 HMG, cyan; Oct6 POU domain, orange. Individual helixes of POU domains are numbered. In both models, the DNA is represented as a light grey surface. Unless indicated otherwise, the representation is kept throughout the manuscript.D. EMSAs with a 2-fold dilutions series starting at 2 μM of the Oct4 POU (left) or the Oct6 POU (right) in the presence of cy5-labeled MORE DNA. The free DNA and DNA bound as monomer or dimer are indicated.E. and F. Bar plots of EMSA-derived cooperativity factors for Oct-Oct homodimers on the MORE (E) or Sox2-Oct heterodimers on the SoxOct element (F) are shown for a panel of six POU proteins. The mean is shown with standard deviation as error bars (n≥3) and Tukey multiple comparison of means was used for assessment of statistical significances (*** p < 0.001, ** p < 0.01, * p < 0.05). N.A.: not assessed.G. Properties of Oct4 and Oct6 homodimers from molecular dynamics (MD) simulations. The structural snapshots illustrate Oct6 (first monomer in orange, second in dark blue) and the 3 parameters used: drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains (excluding the linker); θ = the angle describing the kink in helix α4 (data for both monomers 1 and 2 are shown); d = the distance between the centers of mass of the 2 helices α5; w = the DNA minor groove width in the region between the 2 monomers.


Figure 2A . Zoom - in view of the Oct6 - Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA . Oct6 POUS , orange ; POUHD of the other monomer , blue . Important residues are labelled and Met ( Oct6 ) in position 151 which was identified to guide the DNA - binding preference is marked with a star . Individual helixes of POU domains are numbered . Colour representation as in Fig . 1B , 1C . For clarity , the second monomer of Oct4 / Oct6 is shown in dark blue . B . and C . Network of hydrophobic interactions in Oct6 ( B ) and Oct4 ( C ) . The numbers show the minimal interatomic distances ( excluding hydrogens ) between the residues involved calculated from the simulations . In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged . The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations . D . EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE ( OctOct ) element . The free DNA and DNA bound by monomer or dimer are indicated . E . EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG . The free DNA and DNA bound by monomers or dimer are indicated . Quantifications of cooperativity measurements are shown in Fig . EV3C . F . Bar plot based on single tube EMSAs using cy5 - SoxOct and FAM - MORE DNA elements showing the difference in DNA - binding of WT Oct4 , WT Oct6 , Oct4151M and Oct6151S POU domains . The mean log2 ratio of cy5 ( heterodimer ) and FAM ( homodimer ) band intensities are depicted with standard deviation ( n = 9 ) . Individual data points are shown as gray jitter plots . Tukey multiple comparisons of means was performed to assess significance ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , p > 0 . 05 n . s . ) . Compare to Fig . EV1D and EV3D .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Zoom-in view of the Oct6-Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA. Oct6 POUS, orange; POUHD of the other monomer, blue. Important residues are labelled and Met (Oct6) in position 151 which was identified to guide the DNA-binding preference is marked with a star. Individual helixes of POU domains are numbered. Colour representation as in Fig. 1B, 1C. For clarity, the second monomer of Oct4/Oct6 is shown in dark blue.B. and C. Network of hydrophobic interactions in Oct6 (B) and Oct4 (C). The numbers show the minimal interatomic distances (excluding hydrogens) between the residues involved calculated from the simulations. In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged. The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations.D. EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE (OctOct) element. The free DNA and DNA bound by monomer or dimer are indicated.E. EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG. The free DNA and DNA bound by monomers or dimer are indicated. Quantifications of cooperativity measurements are shown in Fig. EV3C.F. Bar plot based on single tube EMSAs using cy5-SoxOct and FAM-MORE DNA elements showing the difference in DNA-binding of WT Oct4, WT Oct6, Oct4151M and Oct6151S POU domains. The mean log2 ratio of cy5 (heterodimer) and FAM (homodimer) band intensities are depicted with standard deviation (n=9). Individual data points are shown as gray jitter plots. Tukey multiple comparisons of means was performed to assess significance (*** p < 0.001, ** p < 0.01, * p < 0.05, p > 0.05 n.s.). Compare to Fig. EV1D and EV3D.


Figure 3A and B . Molecular models show the position of mutated residues in the structures of two dimers on DNA : Sox2 - Oct4 heterodimer ( A ) and Oct4 - Oct4 homodimer ( B ) . Ser in position 151 ( asterisk ) was mutated to Met in order to shift the preference of Oct4 for homodimerization . The individual helixes of the POU domains are numbered . D . GFP - positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2 , Klf4 , and c - Myc . Colonies were imaged 16 days after second viral infection , using a fluorescence microscope . Scale bars : 250 μm ; objective 10x .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A and B. Molecular models show the position of mutated residues in the structures of two dimers on DNA: Sox2-Oct4 heterodimer (A) and Oct4-Oct4 homodimer (B). Ser in position 151 (asterisk) was mutated to Met in order to shift the preference of Oct4 for homodimerization. The individual helixes of the POU domains are numbered.D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.


Figure 4B . Oct4 - GFP - positive colonies of two O6SKM iPSC lines , expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule ( Oct67K , 22T , LinkO4 , 151S ) . Scale bars : 250 μm . C . Genotyping of two O6SKM iPSC lines . Two stable lines of iPSCs generated with the Oct6 , Sox2 , Klf4 , and c - Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene . OSKM iPSCs and OG2 - MEFs were used as PCR controls . D . Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT - PCR using specific primers . OG2 - MEFs were harvested 5 days after infection and used for comparison as a positive control . E . Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog . Oct4 expression was confirmed by the Oct4 - GFP transgene . DNA stained by DAPI . Scale bars : 100 μm . F . Bisulfite sequencing of genomic Oct4 , Nanog , and Col1a1 promoter regions of O6SKM iPSCs , OSKM iPSCs , and OG2 - MEFs . White and black circles represent unmethylated and methylated CpG sites , respectively . G . Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs ( left ) and O4SK iPSCs ( right ) . Black lines represent a two - fold change in gene expression levels between the paired cell lines . On the right side of the plots , the color bar indicates scattering density . Red and green dots represent up - and downregulated genes , respectively . Positions of selected pluripotency - related genes are highlighted as orange points . H . Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs , illustrating the close relationship between their global gene expression profiles .

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Figure 4B. Oct4-GFP-positive colonies of two O6SKM iPSC lines, expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule (Oct67K,22T,LinkO4,151S). Scale bars: 250 μm.C. Genotyping of two O6SKM iPSC lines. Two stable lines of iPSCs generated with the Oct6, Sox2, Klf4, and c-Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene. OSKM iPSCs and OG2-MEFs were used as PCR controls.D. Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT-PCR using specific primers. OG2-MEFs were harvested 5 days after infection and used for comparison as a positive control.E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm.F. Bisulfite sequencing of genomic Oct4, Nanog, and Col1a1 promoter regions of O6SKM iPSCs, OSKM iPSCs, and OG2-MEFs. White and black circles represent unmethylated and methylated CpG sites, respectively.G. Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs (left) and O4SK iPSCs (right). Black lines represent a two-fold change in gene expression levels between the paired cell lines. On the right side of the plots, the color bar indicates scattering density. Red and green dots represent up- and downregulated genes, respectively. Positions of selected pluripotency-related genes are highlighted as orange points.H. Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.


Figure 5A . In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry : endoderm ( α - fetoprotein , AFP ) , mesoderm ( α - smooth muscle actin , SMA ) , and ectoderm ( α - tubulin , TUJ1 ) . Nuclei ( DNA ) were stained by Hoechst ( blue ) . Scale bars : 200 μm . B . F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice . C . Pictures demonstrating germline transmission ; endogenous Oct4 - GFP signal was detected in the gonads of F1 pups . Scale bars : 200 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm.B. F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice.C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.


figf1 ( a ) HEK293 cells were starved of glucose ( 4 h ) as indicated , endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed . Proteins were resolved by SDS - PAGE and visualized with autoradiography ( top ) or western blotting ( WB ; bottom ) . ( b ) Cells were incubated in glucose - free medium ( 4 h ) as indicated and lysed . Lysates were incubated with lambda phosphatase ( λ PPase ) as indicated . Endogenous Ulk1 mobility was examined by western blotting . ( c ) HA - Ulk1 was transfected into HEK293 cells together with wild - type ( WT ) AMPKα1 or a kinase - dead ( DN ) mutant . Cells were starved of glucose ( 4 h ; Glu ) or amino acids ( - A . A ) and treated with compound C ( 20 μM , C . C ) as indicated . Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting . ( d ) HA - Ulk1 proteins were immunopurified from transfected HEK293 cells , which had undergone glucose starvation ( 4 h ) as indicated . The HA - Ulk1 proteins were treated with λ PPase , and in vitro kinase assays were performed in the presence of GST - ATG13 . Proteins were resolved by SDS - PAGE ; phosphorylated proteins were visualized with autoradiography , HA - Ulk1 by western blotting and GST - Atg13 by Coomassie staining . ( e ) HA - Ulk1 was immunopurified from transfected HEK293 cells under glucose - rich media and treated with AMPK in the presence of cold ATP for 15 min , followed by kinase assays as described in d . In vitro kinase assays were performed in the presence of GST - ATG13 . Proteins were resolved by SDS - PAGE ; phosphorylated proteins were visualized with autoradiography , HA - Ulk1 by western blotting and GST - Atg13 by Coomassie staining . ( f ) AMPK wild - type ( WT ) and α1 / α2 double knockout ( DKO ) MEFs were incubated with or without glucose ( 4 h ) . Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured ( mean ± s . d . , n = 3 ) . Autophosphorylation activity was normalized to Ulk1 protein level ; relative activity is calculated by normalization to Ulk1 activity from AMPK wild - type MEFs in glucose - rich conditions . ( g ) HA - Ulk1 was transfected into HEK293 cells together with vector ( Vec ) or an AMPKα1 kinase - dead mutant ( DN ) . The cells were starved of glucose ( - Glu ) or amino acids ( - A . A ) , or treated with 50 nM rapamycin ( Rapa ) for 3 h before lysis . Left : autophosphorylation activity was assessed and normalized as in f ( mean ± s . d . , n = 3 ) . Right : fold induction in Ulk1 autophosphorylation , compared with Ulk1 autophosphorylation from cells under nutrient - rich conditions . Uncropped images of blots are shown in Supplementary Fig . S5 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom).(b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting.(c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting.(d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.(e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.(f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions.(g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.