SUMOylation Of PML Regulates Dynamic Changes Of PML-NBs In The Cell Cycle

Background:PML nuclear bodies(PML-NBs)are membraneless organelles with diameters ranging from 0.1-1?m in the nucleus of mammalian cells.As a site of protein enrichment in the nucleus,it has been reported that there are more than 200 component proteins in the PML nuclear body.These component proteins are widely involved in transcriptional regulation,cell cycle progression,protein posttranslational modification(PTM),DNA damage repair,virus infection,apoptosis,cell stress...As an important PTM platform,PML-NBs play an essential role in providing SUMOylation environment.Like ubiquitination,SUMOylation is a post-translational modification pathway that exists in eukaryotic cells.In vertebrates,five members of the SUMO family have been reported: SUMO-1,the highly homologous SUMO-2/3,the newly discovered SUMO-4 and SUMO-5.SUMO-2 and SUMO-3 contain an internal consensus site for SUMOylation that is missing in SUMO-1,and thereby the former can form polySUMO chain whereas the latter can only form a monomer.Unlike ubiquitination modification,the SUMOylation usually does not cause degradation of the target protein,but affects its structure and function,such as structural activation,changes in location,and protection from ubiquitin-mediated degradation.Approximately 24% of the proteins in the human proteome have SUMOylation sites,while in the component proteins of PML-NBs,this proportion reaches 48%.DAXX is a typical PMLinteracting protein,which can be SUMOylated and contains a SIM motif.Moreover,it also acts as a chaperone of histone H3.3 and ectopic CENP-A for their deposition into chromosome.CENP-A is a wellknown epigenetically centromere marker,plays an pivotal role in maintaining chromosome stability by recruiting other centromere and kinetochore proteins.It is reported that some CCAN protein can be SUMOylated,whereas PML-NBs is a hotspot for SUMOylation in mammal cell.However,the relationship between PML-NBs and centromere-kinetochore is still poorly studied.A publishment reported that Sp100(a typical PML binding protein)lose its co-localization with PML as cell enter the M phase,another one noted that the mobility of PML-NBs measured by FRAP shows great difference in different cell cycle.Methods: First,we stained PML in He La cells of different cell cycle,and found that the number of PMLNBs in M phase cells are significantly reduced compared with the nuclear body number of interphase cells.We further examined the PML protein level through cell cycle.We observed that the expression level of PML didn't change in different cell cycles.It is speculated that the decrease in the number of PML-NBs results from the increase of the dynamic balance of free/aggregated PML: free PML molecule increases as cell entering M phase,and aggregated PML significantly decreases.We synchronized the He La cells expressing Flag-PML,collect the interphase and metaphase cells respectively,by performing immunoprecipitation and immunoblotting to detect the SUMO conjunction level on PML.In order to study the effect of PML-NBs on the centromere-kinetochore proteins,we used CRISPRi system to knock down the PML and DAXX,respectively in He La,and performed immunofluorescence on different centromere-kinetochore proteins.By using the anti-CENP-B immunofluorescence signals as centromere reference signal,we performed the quantification of fluorescence signals of these centromere-kinetochore proteins.We also explored the assembly mechanism of PML-NBs.By constructing a truncated PML mutant,we determined the key domain of PML to assemble to the nuclear body.Further,we introduced the photolyase homology region(PHR)domain from CRY2 gene of Arabidopsis thaliana.We are establishing and optimizing a light-induced PML-NB assemble system,and further study how PML-NBs kinetics affects mitosis,cell cycle and other cellar process.Results: Our results show that endogenous PML protein level is not changed between interphase and mitotic phase.This data suggests that the dynamic changes(localization pattern and NB number in a cell)of PML-NBs through the cell cycle are not relevant to the PML protein level.The result of immunoprecipitation shows that the level of SUMO modification of PML is slightly reduced in mitotic phase.These data suggests that the level of PML SUMOylation could be the key factor in determining the number of PML-NBs in different cell periods.Our CRISPRi system successfully knocked down the expression of endogenous PML and DAXX,respectively,in He La cells.We performed the quantification of fluorescence intensity of different centromere-kinetochore proteins in specific cell cycle,and found that localization of most kinetochore proteins(8 in 10)we tested are not affected by PML defects.Even those show the positive results(CENP-F and MAD2),the value of T-test is very slight.On the other hand,depletion of DAXX leads to abnormal localization of CENP-A,CENP-C and CENP-F.We constructed a coiled-coil(CC)domain-truncated PML mutant(PML-d CC),and found that PML cannot aggregate and form nuclear bodies after the CC domain is deleted.When we introduce the PHR domain of CRY2(a protein that mediates homo-dimerization after blue light activation)into coiled-coil domain deleted PML construct with 488 nm light stimulation,PML restores its ability of forming nuclear body,but not in that PML-d CC without PHR.Conclusion: The protein level of PML is stable between interphase and mitotic phase,but the level of SUMOylation of PML reduces at mitotic phase comparing with interphase.We reckon that SUMOylation of PML may affect the kinetics of PML-NBs,resulting in a decrease in the number of PML-NBs in M phase.Although PML-NBs are important PTMs platform in the nucleus,PML knockdown does not affect the localization of 8 kinetochore proteins including CCAN protein,central-outer kinetochore proteins,spindle checkpoint proteins,and mitotic kinase to the centromere/kinetochore region.Only the quantification results of CENP-F and MAD2 shows slight difference comparing with control.We can not role out that difference results from statistics deviation.These data are in line with previous studies showing that,pml-/-cell can still keep growth and proliferation,and no obvious phenotype was observed in pml knockout mice.Because defects on kinetochore or mitotic regulatory protein usually lead to lethality.Depletion of DAXX leads to abnormal localization of partial kinetochore proteins including CENP-A,CENP-C and CENP-F in He La cells.Given that DAXX can interact with both CENP-A and CENP-C,we reckon that CENP-F may be a new target of DAXX.And CENP-F also is the only centromere protein that being affected by both DAXX and PML depletion,indicating that it could be the common target of DAXX-PML.But the influence and mechanism of this phenotype needs our further investigation.Currently more than 100 kinetochore proteins are identified,and there are still many other kinetochore proteins we did not analyze in quantification of immunofluorescence signals after PML or DAXX depletion.The homodimerization process mediated by the coiled-coil domain is a prerequisite for the formation of PML-NBs.The deletion of the coiled-coil domain causes the failure of the normal assembly of PML-NBs.Further,we artificially introduced of the PHR domain which mediates dimerization to coiled-coil domain deleted PML restores PML-NBs formation after blue light activation.We will utilize this system to address how the dynamics of PML-NBs regulate mitotic regulators(e.g.,kinetochore proteins),and other cellar process including DNA damage repair,apoptosis,post-translational modification,transcriptional regulation...