Small ubiquitin-like modifiers (SUMO) proteins are small polypeptides that bind to the lysine residues of target proteins to regulate functions. SUMOylation is a post-translational modification that regulates several biological processes such as transcriptional regulation, cell cycle progression, nuclear cytosolic translocation, and DNA damage response. In this study, I found that Zinc Finger Protein 212 (ZNF212) can be SUMOylated by interacting with Protein Inhibitor of Activated STAT 1 (PIAS1) and Protein Inhibitor of Activated STAT3 (PIAS3), which are E3-type SUMO ligases.
Interestingly, the SUMOylation site mutant of ZNF212 was weakly translocated to the DNA damage site and showed hypersensitivity to camptothecin (CPT) treatment compared to the wild type. Combined, these results support the potential for SUMOylation of ZNF212 to regulate the DNA damage response. SENPs: Sentrin-specific proteases KRAB: Kruppel-associated box ZNF212: Zinc finger protein 212 BrdU: 5-bromo-2-deoxyuridine HU: Hydroxyurea.
Background
DNA damage response (DDR)
Small Ubiquitin like Modifier
Like ubiquitination, SUMO modification (SUMOylation) is catalyzed by the cascade enzyme system consisting of SAE1,2 heterodimer as SUMO E1 activating enzyme, E2 SUMO conjugating enzymes UBC9 and E3 ligases including the PIAS family and RanBP2 [13,41]. First, the C-terminal peptide is cleaved from the SUMO precursor to remove some additional amino acids and revealed a di-glycine motif by SENP protease[ 15 ]. The mature SUMO is then bound to the E1 enzyme SAE1/SAE2 and transferred to UBC9 as the E2 conjugating enzyme[16].
The PIAS family and RanBP2 can interact with UBC9 to mediate the transfer of SUMO-conjugated UBC9 to the lysine of a target protein [ 17 ]. SUMO can bind a target protein to inhibit proteasomal degradation by ubiquitination to maintain protein stability. The SUMO processing pathway is conserved in eukaryotes and plays a dynamic role in cell signaling, gene regulation and maintenance of genomic integrity.
Introduction
DNA damage response proteins participate in the DNA damage response through a zinc finger domain [ 28 , 29 ] and several DDR proteins have been modulated by SUMOylation [ 30 – 32 ]. Zinc finger protein 212 (ZNF 212) is a member of a group of Kruppel-associated box (KRAB) domain-containing zinc finger proteins known as highly conserved mammalian transcription factors [ 33 ]. Recently, our laboratory identified ZNF212 as a novel interaction partner with TRAIP that contributes to genomic integrity in cooperation with TRAIP [ 34 ].
Here, I propose that ZNF212 may participate in the DNA damage response through post-translational modifications. In this study, I identified PIAS1 and PIAS3 as novel binding partners for ZNF212 and confirmed that ZNF212 was modified by SUMO at the head site lysine 217. This study supports the possibility that SUMOylation of ZNF212 contributes to proper DNA damage response.
Materials and methods
Transformant selection was performed by growth with Trp, Leu, His, and Ade solid media containing 30 mM 3-aminotriazole (3-AT). Hela cells were transfected twice with control and ZNF212 siRNAs at 24 hour time intervals in 60mm dishes. 24 h after second transfection, cells were transfected with Myc-tagged ZNF212 wild-type and K217R expression plasmid.
Seven days after CPT treatment, cells were cleaned with PBS, fixed with 4% paraformaldehyde, and stained with 2% methylene blue.
Results
To investigate whether PIAS1 and PIAS3 affect ZNF212 SUMOylation, Myc-ZNF212 was transfected with or without FLAG-PIAS1 and PIAS3 into HEK293T cells. The results showed that PIAS1 and PIAS3 promote the SUMOylation of ZNF212 (Figure 6A). The increased expression level of the Myc-ZNF212 SUMOylated band is dependent on the expression of FLAG-PIAS1 and FLAG-PIAS3. To determine which sites of ZNF212 should be SUMOylated, the SUMOylation sites were predicted using the SUMOplt, SUMOsp and seeSUMO analysis programs (Figure 7A).
Since many studies have reported that the protein modified by SUMO is regulated in the DDR, I confirmed whether SUMOylation of ZNF212 is also involved in DDR through DNA-damaging agents. I confirmed that ZNF212 modified by SUMO induced by DNA damage in previous data, I used K217A as SUMOylation-defective ZNF212 to detect the function of SUMOylation in response to DNA damage. To determine whether ZNF212 is translocated to the DNA damage site and its correlation with SUMOylation, I applied the microirradiation system with 355nm UVA to measure the accumulation of GFP-ZNF212 WT and K217A with 5-bromo-2-deoxyuridine (BrdU) treatment investigation.
These data suggest that ZNF212 SUMOylation may affect translocation activity to sites of DNA damage. Based on the potential for ZNF212 to play a biological function in DDR, I examined the effects of ZNF212 depletion on clonogenic survival of HeLa cells in response to CPT treatment on induced DNA damage. Taken together, SUMOylation of ZNF212 may facilitate translocation to DNA damage sites and play an important role for cell survival in DNA damage response.
List of potential interacting partners of ZNF212 and PIAS1, PIAS3 by yeast two-hybrid target analysis. c), (d). The C terminus of ZNF212 interacts with PIAS1 and PIAS3. a) Schematics of ZNF212 deletion constructs. b), (c) Immunoprecipitation analysis to determine the PIAS1 and PIAS3 interaction region in ZNF212. HEK293T cells were co-transfected with Myc-tagged ZNF212 and FlAG-tagged PIAS1 or PAIS3 constructs. a) Myc-ZNF212 was transfected into HEK293T cells and the cell lysate was examined by immunoblotting with anti-Myc antibody.
ZNF212 is SUMOylated by PIAS1 and PIAS3. a) HEK293T cells were co-transfected with Myc-ZNF212 and FLAG tagged PIAS family including PIAS1, PIAS3 and PIASy. K217 is the major SUMOylation site of ZNF212. a) SUMOylation sites of ZNF212 were predicted by the SUMOplot™, SeeSUMO and SUMOsp SUMO binding site prediction analysis programs. K217 is the major SUMOylation site of ZNF212. d) Myc-ZNF212 WT or point mutant expression vector was transfected into HEK293T cell.
After 24 h, transfected cell lysates were examined using immunoblotting with anti-Myc antibody. e) Myc-ZNF212 wild-type and SUMOylation site mutants K217A and K217R were co-transfected into HEK293T cells with HA-SUMO1 or HA-SUMO2. Then cell lysates are analyzed by immunoblotting. SUMOylation involved in recruitment of ZNF212 to DNA damage site. a) Dynamic recruitment of GFP-ZNF212 WT or K217A to DNA damage sites in U2OS cells using UVA laser microirradiation.
Discussion
I also discovered that ZNF212 was recruited to DNA damage site upon laser microirradiation and hypothesized that ZNF212 would function in DNA damage response. Furthermore, SUMOylation is well known to modulate DNA-binding activity [ 45 ], so it is necessary to confirm whether it modulates the DNA-binding activity of ZNF212. ZNF212 also participates in ICL repair, whether SUMOylation occurs in response to ICL damage needs to be investigated in a further study. In addition, it is necessary to confirm which SUMO paralogs are fixed by PIAS1 and PIAS3 at distinct DNA lesions.
Also, other SUMOylation enzymes, such as UBE2I and SENP2, have been identified using yeast two-hybrid screening, and it is also necessary to determine how deSUMOylation enzymes or E2 ligases affect ZNF212 modification. Since there are many reports that protein-protein interactions are regulated by SUMOylation, ZNF212 also participates in DNA damage through its interaction with TRAIP, suggesting that SUMOylation of ZNF212 may directly or indirectly modify this protein interaction. Although not defined here, as the functions regulated by SUMO are inexhaustible, this study may support the possibility that the DNA damage response is controlled by SUMOylation.
Corrigendum, I discovered that SUMOylation of ZNF212 contributes to translocate to the site of damage and sustain cell survival to DNA damage-inducing agent. Further studies on how ZNF212 participates in DNA damage responses should be performed, and understanding more detailed mechanisms is essential to determine cellular regulation by SUMOylation of ZNF212 in DNA damage response.
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Acknowledgement
