I find that ZNF212 colocalizes with TRAIP at sites of DNA damage and that the recruitment of TRAIP or ZNF212 to sites of DNA damage is interdependent. ZNF212 promotes genomic integrity by interacting with TRAIP Figure 2.1 Identification of ZNF212 from yeast two-hybrid assay with TRAIP.
PWWP2B promotes DNA end resection and homologous recombination
Introduction
1-1. DNA damage response
1-2. Various type of DNA damage and repair pathway
1-3. Interstrand crosslink repair
1-4. Double strand break repair
1-5. Gastric cancer
1-6. References
Park, Y.; Ki, M., Population attributable fraction of helicobacter pylori infection-related gastric cancer in Korea: a meta-analysis. C.; Passalacqua, R.; Valeri, N., Microsatellite instability in gastric cancer: molecular bases, clinical perspectives and new treatment approaches.
ZNF212 promotes genomic integrity through interaction with TRAIP
2-1. Introduction
TRAIP appears to function as a master regulator that modulates the choice of ICL repair pathways by regulating the status of MCM7 ubiquitination in Xenopus egg extracts 17 . However, how TRAIP modulates damage signaling, HR and ICL repair is not fully understood.
2-2. Materials and Methods
Thirty-five metaphase cells were randomly sampled in the indicated condition for a statistical analysis (unpaired t test). The cross-linking reaction was quenched using 0.125 M glycine, and then the cells were washed three times with PBS.
2-3. Results
In HeLa cells expressing GFP-TRAIP and mCherry-ZNF212, both proteins were recruited to the sites of DNA damage within 1 minute of laser microirradiation (Figure 2.7A). I found that depletion of ZNF212 results in moderately increased sensitivity to MMC, HU and CPT compared to HeLa cells treated with control siRNA (Figure 2.9B - D). Therefore, I performed SCE assay in HeLa cells and found that depletion of ZNF212 resulted in a significant decrease of SCE compared to cells treated with control siRNA (Figure 2.10D).
Additionally, I generated Zfp212 and Traip (ZT-DKO) double knockout cells and confirmed sensitivity to MMC and CPT (Figure 2.18A). As expected, Zfp212 is epistatic to Traip for DNA damage repair induced by MMC or CPT (Figure 2.18B and C). I found that deletion of the Traip gene or the Neil3 gene resulted in hypersensitivity to TMP-induced DNA damage (Figure 2.21A).
Furthermore, as shown in Figure 2.21C, deletion of the Neil3 gene in FancbKO mESCs led to increased sensitivity to TMP-ICL. I found that ZNF212 and NEIL3 were recruited to DNA lesions induced by both TMP or angelicin (Figures 2.27A and 2.27B) in U2OS cells. Both GFP-NEIL3 and mCherry-ZNF212 were found to localize to and then dissociate from DNA damage sites with similar kinetics (Figure 2.28E).
I found that depletion of ZNF212 impaired NEIL3 translocation to sites of DNA damage in the presence of TMP but not in the presence of angelicin (Figures 2.29A and 2.29B).
2-4. Conclusion and Discussion
The immunoprecipitation data indicate that N- and C-terminus of ZNF212 domains are associated with TRAIP (Figure 2.2C). However, as shown in Figure 2.6A, the fluorescent intensity of ZNF212-N and ZNF212-C stripes was relatively lower compared to that of ZNF212 wild type although ZNF212-WT, ZNF212-N and ZNF212-C accumulated on the laser stripes in most of the cells. Further studies showed that the ZNF212-ΔM showed similar level of recruitment to that of ZNF212-WT, suggesting that both N- and C-terminus of ZNF212 are required for full recruitment of TRAIP to the sites of DNA damage ( Figure 2.6A).
Interestingly, however, expression of ZNF212-N or ZNF212-C in cells depleted of endogenous ZNF212 rescues sensitivity to MMC, CPT and HU (Figure 2.9B-D) and also impairs RAD51 and RPA2 foci formation at the level of ZNF212-WT. (Figure 2.10A and B). Together with the lower recruitment of ZNF212-N and ZNF212-C to sites of DNA damage, these findings suggest that having partial TRAIP activity may be sufficient to play a specific role in the DNA damage response. DNA and DNA repair. Specifically, TRAIP appears to interact with either ZNF212 or RNF20/40 through the same domain (D5 in Figure 2.2A).
In addition, TRAIP appears to function as an important factor for ICL repair as a regulatory factor upstream of both the NEIL3 and FA pathways in mESC lines. These results together with mESC lines used in this study will be informative to understand the molecular basis of the ICL repair pathways in detail (Figure 2.32).
2-5. References
S.; Choi, Y.; Rho, J., Early embryonic lethality caused by targeted disruption of the TRAF-interacting protein (TRIP) gene. Kim, H.; Huang, J.; Chen, J., CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response. Y.; Dodds, S.; Hu, L.; Luo, G.; Hasty, P., RECQL5 and BLM exhibit divergent functions in cells defective for the Fanconi anemia pathway.
W.; Dungrawala, H.; Cortez, D., Functional analysis of the replication fork proteome identifies BET proteins as PCNA regulators.
PWWP2B promotes DNA end resection and homologous recombination
3-1. Introduction
During HR, the MRE11-RAD50-NBS1 (MRN) complex first recognizes DSBs and then initiates DNA end resection that generates 3' single-stranded DNA (ssDNA) overhangs. For DNA end resection, CtIP was found to localize to the sites of DSBs and associate with the MRN complex. CtIP at DSB sites facilitates DNA end resection by promoting the nuclease activity of the MRN complex.
It was shown that UHRF1 was sufficient to channel recovery to the HR pathway in S phase, while both UHRF1 and CtIP were required for this process in G2 phase. /or DNA repair pathways. Recently, it was reported that the PWWP2B protein together with PWWP2A formed a complex with NuRD subunits such as MTA1/2/3, HDAC1/2 and RBBP4/7 and played an important role in the regulation of histone acetylation and gene expression 20 .
I also showed that PWWP2B depletion severely impaired IR-induced BRCA1, RPA2, and RAD51 foci formation, suggesting that PWWP2B promoted DNA end resection in the HR-mediated repair process. In summary, our findings identified PWWP2B as a novel DDR factor that interacted with UHRF1 and promoted HR-mediated DSB repair by facilitating DNA end resection.
3-2. Materials and Methods
For immunoprecipitation, the cells were washed with ice-cold phosphate-buffered saline (PBS) and then in NETN buffer (0.5%. Forty-eight hours after the second transfection, the transfected cells were irradiated at the indicated doses using a radiation source) Fourteen days after irradiation, the cells were washed with PBS, fixed, stained with 2% (w/v) methylene blue, and the colonies were counted.
The cell lysates of the HEK293T cells transfected with plasmids encoding the indicated proteins were incubated with the GST fusion protein or GST for 1 h at 4°C. U2OS DR-GFP or EJ5-GFP cells were first treated with the indicated siRNAs in 24-well plates and then transfected with 0.4 μg of I-SceI per well 24 h after siRNA transfection. Seventy-two hours after I-SceI transfection, cells were trypsinized and the percentage of GFP-positive cells was determined by flow cytometry.
Quantitative analysis of end resection using ER-AsiSI U2OS cells was performed as previously described 23. The percentage of ssDNA generated by resection at the selected sites was calculated as previously described (Zhou et al, 2014).
3-3. Results
Endogenous immunoprecipitation of PWWP2B-interacting proteins further demonstrated that PWWP2B is associated with UHRF1 (Figure 3.2C). Taken together, these data indicated that UHRF1 was associated with the PWWP2B protein via the UHRF1 binding domain (UBD) (Figure 3.4D). As a result, I found that both GFP-UHRF1 and mCherry-PWWP2B rapidly translocated to DNA damage sites within 1 minute of laser microirradiation (Figure 3.5E).
However, GFP-PWWP2B D2 and D3 barely localized to the laser stripes, indicating that the PWWP2B domains responsible for UHRF1 interaction were critical for PWWP2B localization to sites of DNA damage (Figure 3.6A). First, I found that damage-induced γH2AX foci disappeared slowly in PWWP2B-depleted HeLa cells after ionizing radiation (IR) compared to cells treated with control siRNA (Figure 3.9A). Reconstitution of PWWP2B WT into PWWP2B-KD cells successfully restored IR-induced RAD51 foci formation (Figure 3.10B).
Remarkably, PWWP2B depletion made the cells more sensitive to IR compared to the control KD cells (Figure 3.10E). Interestingly, the depletion of PWWP2B leads to severe decreases in RPA phosphorylation in response to DNA damage induced by IR (Figure 3.11B).
3-4. Conclusion and Discussion
In addition to its roles in transcriptional regulation, H3K36 methylation has been shown to be a critical chromatin component of the DDR and associated with DNA repair in the regions of active transcription 32 - 33 . PWWP2B was associated with H3K36me3 at actively transcribed genes 20 and important for HR-mediated DNA repair (Figures 3.9 and 3.10), raising the possibility that PWWP2B plays roles in DDR and DSB repair in the regions of active genes through regulation of chromatin status modification. I found that the PWWP2B UBD domain responsible for the interaction with UHRF1 was critical for the functions of PWWP2B in DDR and HR-mediated DSB repair in response to IR (Figures 3.9 and 3.10).
Interestingly, the UBD domain alone was sufficient for translocation to sites of DNA lesions after microirradiation (Figure 3.6B), highlighting the functional importance of the interaction between PWWP2B and UHRF1 in HR-mediated DDR and DSB repair. In addition to the interaction regions (UBD aa), I found that the adjacent region also played an important role in the formation of RAD51 foci and cell survival in response to IR. I showed that PWWP2B interacted with UHRF1 and that the interaction was critical for its recruitment.
I also showed that PWWP2B participates in DDR and HR-mediated DSB repair by interacting with UHRF1, a known important factor for the choice of the DDR and DSB repair pathway. pathway and HR-mediated DSB repair, but also its importance in clinical settings, especially in the prognosis of cancer patients based on mutation status and mRNA expression level.
3-5. References
H.; Lou, Z.; Pei, H., A cell cycle-dependent BRCA1-UHRF1 cascade regulates DNA double-strand break repair pathway choice. Qiu, C.; Sawada, K.; Zhang, X.; Cheng, X., The PWWP domain of the mammalian DNA methyltransferase Dnmt3b defines a novel family of DNA-binding folds. M.; van Ingen, H., Nucleosomal DNA binding directs recognition of H3K36 methylated nucleosomes by the PSIP1-PWWP domain.
Ouyang, H.; Furukawa, T.; Abe, T.; Kato, Y.; Horii, A., The apoptosis promoter BAX gene is mutated in genetically unstable colorectal, gastric, and endometrial cancers. T.; Cano, D.; Böttcher, R.; van der Korput, H.; Dinjens, W.; Jenster, G.; Trapman, J., A mononucleotide repeat in PRRT2 is an important and frequent target of mismatch repair deficiency in cancer.
Acknowledgements
