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Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Ziv O, Zeisel A, Mirlas-Neisberg N, Swain U, Nevo R, Ben-Chetrit N, Martelli MP, Rossi R, Schiesser S, Canman CE, Carell T, Geacintov NE, Falini B, Domany E, Livneh Z - Nat Commun (2014)

Bottom Line: We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη.Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη.These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

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The NPM1c+ mutation causes DNA polη degradation and TLS deficiency.(a) Immunoblot of polη extracted from OCI/AML2 and OCI/AML3 cells expressing wt NPM1 and NPM1c+ alleles, respectively. (b) qPCR of POLH and NPM1 mRNA levels from OCI/AML2 and OCI/AML3 cells. Mean values±s.e.m. of three replicas are presented. (c) TLS in OCI/AML2 and OCI/AML3 cells. TLS across a site-specific TT CPD using the gapped plasmid-based, colony-forming TLS assay is shown. Mean values±s.e.m. of three replicas are presented, two-tailed t-test, P value <0.003). Mutagenicity was addressed by sequencing 96 TLS events per cell line, χ2-test, P value <0.01. (d) Proteasomal inhibition rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with MG132 for the indicated time to inhibit proteasomal activity. Tubulin served as a loading control. (e) NPM1 nuclear retention rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with Leptomycin B (LMB) for the indicated time or its solvent as a control. Tubulin served as a loading control. (f–k) Density plots and histograms representing fluorescence-activated cell sorting analysis of HEK293 cells stably expressing GFP-polη, and partially transfected with mCherry-tagged NPMc+ (f,g), wt NPM1 (h,i) and control mCherry (j,k). Data from mCherry-negative (untransfected) cells in (g,i,k) are in red and correspond to the left y axis, while that from mCherry-positive (transfected) cells are in blue and correspond to the right y axis. All blots are representative of three independent experiments. siCont, control siRNA.
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f6: The NPM1c+ mutation causes DNA polη degradation and TLS deficiency.(a) Immunoblot of polη extracted from OCI/AML2 and OCI/AML3 cells expressing wt NPM1 and NPM1c+ alleles, respectively. (b) qPCR of POLH and NPM1 mRNA levels from OCI/AML2 and OCI/AML3 cells. Mean values±s.e.m. of three replicas are presented. (c) TLS in OCI/AML2 and OCI/AML3 cells. TLS across a site-specific TT CPD using the gapped plasmid-based, colony-forming TLS assay is shown. Mean values±s.e.m. of three replicas are presented, two-tailed t-test, P value <0.003). Mutagenicity was addressed by sequencing 96 TLS events per cell line, χ2-test, P value <0.01. (d) Proteasomal inhibition rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with MG132 for the indicated time to inhibit proteasomal activity. Tubulin served as a loading control. (e) NPM1 nuclear retention rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with Leptomycin B (LMB) for the indicated time or its solvent as a control. Tubulin served as a loading control. (f–k) Density plots and histograms representing fluorescence-activated cell sorting analysis of HEK293 cells stably expressing GFP-polη, and partially transfected with mCherry-tagged NPMc+ (f,g), wt NPM1 (h,i) and control mCherry (j,k). Data from mCherry-negative (untransfected) cells in (g,i,k) are in red and correspond to the left y axis, while that from mCherry-positive (transfected) cells are in blue and correspond to the right y axis. All blots are representative of three independent experiments. siCont, control siRNA.

Mentions: The C terminus of NPM1 is mutated in ~30% of all AML patients, resulting in loss of the nucleolar localization signal and the generation of a de novo nuclear export signal. This leads to mislocalization of mutant NPM1 (designated NPM1c+), and partially of wild-type (wt) NPM1 interacting with it, in the cytoplasm4043. These mutations strongly correlate with better response of patients to chemotherapy and better clinical outcome4445. Because of the role of NPM1 in stabilizing polη in the nucleus, we hypothesized that mislocalization of NPM1 to the cytoplasm might cause destabilization of polη, similar to the effect that we have observed in cells in which NPM1 was knocked down. To this end, we examined polη levels in OCI/AML2 and OCI/AML3 cell lines derived from AML patients, either without or with the NPM1c+ mutation, respectively. As can be seen in Fig. 6a, the amount of polη was much lower in the NPM1c+ AML cell line compared with the NPM1wt AML cell line, whereas the POLH mRNA levels were similar (Fig. 6b). Consistently with the lower polη amount, TLS across a TT CPD was lower (two-tailed t-test, P value <0.003), and more mutagenic (χ2-test, P value <0.01) in the NPM1c+ AML cells compared with the NPM1wt AML cells (Fig. 6c). Proteasome inhibition using MG132 rescued the level of polη in the NPM1c+ AML cell line (Fig. 6d), indicating that polη is subjected to excessive proteasomal degradation, as was the case in cells in which NPM1 was knocked down. To further support the notion that it is the mislocalization of NPM1 in the AML cells that causes the polη deficiency, we treated the cells with Leptomycin B, an inhibitor of nuclear export via exportin 1, previously shown to prevent the exclusion of NPM1 from the nucleus46. As can be seen in Supplementary Fig. 8, NPM1 was present mainly in the cytoplasm in NPM1c+ AML cells, but not in the NPM1wt AML cells, as expected. Upon addition of Leptomycin B, The NPM1c+ AML cells exhibited nuclear localization of NPM1, similarly to the NPM1wt cells. Examining the level of polη under these conditions revealed that Leptomycin B treatment did not change the amount of polη in NPM1wt AML cells, but caused a significant increase in polη in the NPM1c+ AML cells (Fig. 6e), consistent with a role of NPM1 in stabilizing polη in the nucleus. Furthermore, ectopic expression of mCherry-NPM1c+ in HEK293 cells that stably express GFP-polη caused a significant twofold decrease in the amount of GFP-polη as indicated by flow cytometry analysis (Fig. 6f,g). This effect was not observed when mCherry-NPM1wt or mCherry alone were expressed (Fig. 6h–k). Taken together, these results establish a role for NPM1 in the regulation of TLS, and suggest that the prevalent NPM1c+ mutation results in reduced levels of polη, thereby leading to defective TLS.


Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Ziv O, Zeisel A, Mirlas-Neisberg N, Swain U, Nevo R, Ben-Chetrit N, Martelli MP, Rossi R, Schiesser S, Canman CE, Carell T, Geacintov NE, Falini B, Domany E, Livneh Z - Nat Commun (2014)

The NPM1c+ mutation causes DNA polη degradation and TLS deficiency.(a) Immunoblot of polη extracted from OCI/AML2 and OCI/AML3 cells expressing wt NPM1 and NPM1c+ alleles, respectively. (b) qPCR of POLH and NPM1 mRNA levels from OCI/AML2 and OCI/AML3 cells. Mean values±s.e.m. of three replicas are presented. (c) TLS in OCI/AML2 and OCI/AML3 cells. TLS across a site-specific TT CPD using the gapped plasmid-based, colony-forming TLS assay is shown. Mean values±s.e.m. of three replicas are presented, two-tailed t-test, P value <0.003). Mutagenicity was addressed by sequencing 96 TLS events per cell line, χ2-test, P value <0.01. (d) Proteasomal inhibition rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with MG132 for the indicated time to inhibit proteasomal activity. Tubulin served as a loading control. (e) NPM1 nuclear retention rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with Leptomycin B (LMB) for the indicated time or its solvent as a control. Tubulin served as a loading control. (f–k) Density plots and histograms representing fluorescence-activated cell sorting analysis of HEK293 cells stably expressing GFP-polη, and partially transfected with mCherry-tagged NPMc+ (f,g), wt NPM1 (h,i) and control mCherry (j,k). Data from mCherry-negative (untransfected) cells in (g,i,k) are in red and correspond to the left y axis, while that from mCherry-positive (transfected) cells are in blue and correspond to the right y axis. All blots are representative of three independent experiments. siCont, control siRNA.
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Related In: Results  -  Collection

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f6: The NPM1c+ mutation causes DNA polη degradation and TLS deficiency.(a) Immunoblot of polη extracted from OCI/AML2 and OCI/AML3 cells expressing wt NPM1 and NPM1c+ alleles, respectively. (b) qPCR of POLH and NPM1 mRNA levels from OCI/AML2 and OCI/AML3 cells. Mean values±s.e.m. of three replicas are presented. (c) TLS in OCI/AML2 and OCI/AML3 cells. TLS across a site-specific TT CPD using the gapped plasmid-based, colony-forming TLS assay is shown. Mean values±s.e.m. of three replicas are presented, two-tailed t-test, P value <0.003). Mutagenicity was addressed by sequencing 96 TLS events per cell line, χ2-test, P value <0.01. (d) Proteasomal inhibition rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with MG132 for the indicated time to inhibit proteasomal activity. Tubulin served as a loading control. (e) NPM1 nuclear retention rescued Polη deficiency in NPM1c+ AML cells. Polη levels in OCI/AML2 and OCI/AML3 cells pretreated with Leptomycin B (LMB) for the indicated time or its solvent as a control. Tubulin served as a loading control. (f–k) Density plots and histograms representing fluorescence-activated cell sorting analysis of HEK293 cells stably expressing GFP-polη, and partially transfected with mCherry-tagged NPMc+ (f,g), wt NPM1 (h,i) and control mCherry (j,k). Data from mCherry-negative (untransfected) cells in (g,i,k) are in red and correspond to the left y axis, while that from mCherry-positive (transfected) cells are in blue and correspond to the right y axis. All blots are representative of three independent experiments. siCont, control siRNA.
Mentions: The C terminus of NPM1 is mutated in ~30% of all AML patients, resulting in loss of the nucleolar localization signal and the generation of a de novo nuclear export signal. This leads to mislocalization of mutant NPM1 (designated NPM1c+), and partially of wild-type (wt) NPM1 interacting with it, in the cytoplasm4043. These mutations strongly correlate with better response of patients to chemotherapy and better clinical outcome4445. Because of the role of NPM1 in stabilizing polη in the nucleus, we hypothesized that mislocalization of NPM1 to the cytoplasm might cause destabilization of polη, similar to the effect that we have observed in cells in which NPM1 was knocked down. To this end, we examined polη levels in OCI/AML2 and OCI/AML3 cell lines derived from AML patients, either without or with the NPM1c+ mutation, respectively. As can be seen in Fig. 6a, the amount of polη was much lower in the NPM1c+ AML cell line compared with the NPM1wt AML cell line, whereas the POLH mRNA levels were similar (Fig. 6b). Consistently with the lower polη amount, TLS across a TT CPD was lower (two-tailed t-test, P value <0.003), and more mutagenic (χ2-test, P value <0.01) in the NPM1c+ AML cells compared with the NPM1wt AML cells (Fig. 6c). Proteasome inhibition using MG132 rescued the level of polη in the NPM1c+ AML cell line (Fig. 6d), indicating that polη is subjected to excessive proteasomal degradation, as was the case in cells in which NPM1 was knocked down. To further support the notion that it is the mislocalization of NPM1 in the AML cells that causes the polη deficiency, we treated the cells with Leptomycin B, an inhibitor of nuclear export via exportin 1, previously shown to prevent the exclusion of NPM1 from the nucleus46. As can be seen in Supplementary Fig. 8, NPM1 was present mainly in the cytoplasm in NPM1c+ AML cells, but not in the NPM1wt AML cells, as expected. Upon addition of Leptomycin B, The NPM1c+ AML cells exhibited nuclear localization of NPM1, similarly to the NPM1wt cells. Examining the level of polη under these conditions revealed that Leptomycin B treatment did not change the amount of polη in NPM1wt AML cells, but caused a significant increase in polη in the NPM1c+ AML cells (Fig. 6e), consistent with a role of NPM1 in stabilizing polη in the nucleus. Furthermore, ectopic expression of mCherry-NPM1c+ in HEK293 cells that stably express GFP-polη caused a significant twofold decrease in the amount of GFP-polη as indicated by flow cytometry analysis (Fig. 6f,g). This effect was not observed when mCherry-NPM1wt or mCherry alone were expressed (Fig. 6h–k). Taken together, these results establish a role for NPM1 in the regulation of TLS, and suggest that the prevalent NPM1c+ mutation results in reduced levels of polη, thereby leading to defective TLS.

Bottom Line: We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη.Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη.These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

Show MeSH
Related in: MedlinePlus