SUMO E3 ligase CBX4 regulates hTERT-mediated transcription of CDH1 and promotes breast cancer cell migration and invasion

hTERT, the catalytic component of human telomerase enzyme, is regulated by post-translational modifications, like phosphorylation and ubiquitination by multiple proteins which remarkably affects the overall activity of the enzyme. Here we report that hTERT gets SUMOylated by SUMO1 and polycomb protein CBX4 acts as the SUMO E3 ligase of hTERT. hTERT SUMOylation positively regulates its telomerase activity which can be inhibited by SENP3-mediated deSUMOylation. Interestingly, we have established a new role of hTERT SUMOylation in repression of E-cadherin gene expression and consequent triggering on the epithelial-mesenchymal-transition (EMT) program in breast cancer cells. We also observed that catalytically active CBX4, leads to retention of hTERT/ZEB1 complex onto E-cadherin promoter leading to its repression through hTERT-SUMOylation. Further through wound healing and invasion assays in breast cancer cells, we showed the tumour promoting ability of hTERT was significantly compromised upon overexpression of SUMO-defective mutant of hTERT. Thus our findings establish a new post-translational modification of hTERT which on one hand is involved in telomerase activity maintenance and on the other hand plays a crucial role in regulation of gene expression thereby promoting migration and invasion of breast cancer cells.


Introduction
Telomeres are the repetitive, non-coding hexameric DNA sequences that protect linear chromosomal end from degradation and inappropriate processing as damaged DNA. Despite this protective role, telomere shortening occurs in the normal somatic cells due to intrinsic limitations of the replication machinery, and this shortening causes cellular senescence. However, cancer cells are able to avoid this shortening and thereby senescence by activating the ribonucleoprotein enzyme telomerase, which can extend telomeres. The human telomerase enzyme contains three essential components necessary for its function [1] : telomerase reverse transcriptase (hTERT), the catalytic subunit which is an RNA dependent DNA polymerase; the telomerase RNA component (hTERC), which acts as a template for the enzyme telomerase to synthesize the telomeric DNA; and the protein dyskerin which plays acrucial role in maintaining the stability of hTERC. Several epigenetic modifications in cancer cells have been reported to be critically important for preserving telomeric integrity. For example, methylation status of the subtelomeric DNA repeats is reported to be inversely correlated with telomere length and telomere recombination in various human cancers [2], and histone methylation on the promoter of hTERT was also found to control the catalytic activity of human telomerase [3]. Additionally, several studies have highlighted the importance of post-translational modifications (PTMs) of telomeric proteins in maintaining the telomere length and telomeric integrity. SUMOylation is one such PTM which has been reported to be important in telomere maintenance. Studies in different yeast mutants revealed that SUMOylation of different telomere associated proteins like Yku70/80, Sir4, Sir2 and Cdc13 limits the accessibility of telomeres to telomerase and SUMO-defective mutants effectively show abnormally lengthened telomeres [4,5]. In humans, crosstalk between SUMOylation and ubiquitination is needed for the stability of an important member of shelterin complex, TRF2 [6]. Another important protein of the human telomerase enzyme complex, dyskerin also gets SUMOylated and disruption of dyskerin SUMOylation leads to impaired telomerase activity and eventually causes a fatal premature ageing syndrome, dyskeratosis congenital (DKC) [7]. Furthermore, the role of SUMOylation has also been well studied for maintaining telomere length in the cells which follow the Alternative lengthening of telomeres (ALT) pathway for telomere elongation. For the formation of ALT-associated PML bodies Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200359/893052/bcj-2020-0359.pdf by guest on 20 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200359 (APB) and the localization of telomeres into it, the SUMOylation of TRF1 and TRF2 by SUMO E3 ligase MMS2 found to be an indispensable requirement [8].
Polycomb group (PcG) complex is a well known transcriptional repressor complex that regulates the expression of the target genes either by direct repression of the transcription machinery or by regulating the higher-order chromatin structures. Interestingly, earlier studies have indicated an association of polycomb group of proteins with telomere maintenance. In Drosophila, PcG proteins were found to bind with telomeric associated sequences (TASs) taking significant part in telomeric position effect (TPE) [9]. BMI1, an important polycomb group member and wellstudied oncogene, was found to induce telomerase enzyme and helped in immortalization of the human mammary epithelial cells [10]. Correlation between BMI1 and telomerase activity was also observed in case of human ovarian cancer [11] and as well as in hTERT induced epithelial to mesenchymal transition (EMT) of oral epithelial cells [12]. Our protein of interest, CBX4 (or Pc2) is also an integral component of canonical polycomb repressive complex 1 (cPRC1). It was discovered as a SUMO E3 ligase which enhances the SUMOylation status of the transcriptional co-repressor CtBP in both in vitro and ex vivo context by recruiting the substrate protein and the E2, Ubc9 within the PcG body [13]. Later-studies demonstrated that CBX4 can SUMOylate several other proteins like BMI1 itself, HNRNPK, HIPK2 etc. and recruit them at the DNA damage site by initiating p53 mediated DNA damage response (DDR) pathway [14][15][16]. Being a polycomb group member, the role of CBX4 has been well documented in transcriptional repression. CBX4-mediated SUMOylation of 'de novo' methyltransferase Dnmt3a and zinc finger protein CTCF contributes significantly to their repressive activity [17,18].
CBX4 mostly acts as a tumor promoting gene in hepatocellular carcinoma and osteosarcoma by modulating several biological pathways through its SUMO E3 ligase activity [17][18][19]. But evidences suggested that it might also exert non-canonical function as a tumor-supressor in colorectal carcinoma independent of its SUMO E3 ligase, chromodomain and polycomb complex activity [22]. Recently the role of this protein has also been shown in breast cancer through activation of Notch 1 signaling pathway mediated by miR-137 [20]. Although, the individual roles of CBX4 and telomerase have been well-studied in the context of cancer progression, to our knowledge, there has been no report of a functional association between the above two proteins. The current study investigates the role of SUMO E3 ligase CBX4 in the regulation of telomerase activity and for the first time we show that hTERT gets SUMOylated by Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200359/893052/bcj-2020-0359.pdf by guest on 20 September 2020 6 SUMO1 and CBX4 acts as the SUMO E3 ligase of hTERT. Fascinatingly, we found that CBX4 mediated SUMOylation of hTERT does not only alter the telomerase activity but also plays an important role in hTERT/ZEB1 mediated transcriptional regulation of E-cadherin and in turn epithelial-to-mesenchymal (EMT) progression. Further in depth analysis has revealed the epigenetic significance and relevance of this regulation in the context of breast cancer. Tokyo, Japan) using 100x objective.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as described elsewhere [27]. Briefly, the cells were crosslinked with 1% formaldehyde and then 0.125M Glycine was added to stop the reaction. Cells  subjected to qPCR analysis using gene specific primers mentioned in Table 2.

RNA Extraction & Quantitative real time PCR (qRT-PCR)
Total cellular RNA was extracted using TRIZOL reagent (Thermo Fisher Scientific,Waltham, MA, USA) following manufacturer's instructions and RNA quantity, quality was determined Data points were fitted to a linear regression plot and from that arbitrary telomerase units were calculated relative to the TSR8 amplification.

Wound healing assay
Wound healing assay was performed as described elsewhere [29]. Briefly, MDA-MB-231 cells were seeded in a 6-well dish and at around 80% confluency, cells were transfected with indicated plasmids. After 24 hours, cell surface was scratched with a 200 μl sterile tip and this time point was mentioned as 0hr. Images were captured with Nikon T1 E100 microscope (Nikon, Minato, Tokyo, Japan) at 0hr, 24hrs and 48 hrs (post-scratch) to determine the wound healing by the cells reflecting their migratory ability. The images were further analyzed using Image J software (NIH, Bethesda, Maryland, USA) to quantify the percentage of wound recovery by the cells.
Statistical analysis were done based on three replicate experiments.

Transwell invasion assay
Transwell chambers (Corning Inc., Corning, New York, USA) coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were used to perform invasion assay following the protocol as delineated elsewhere [29]. In brief, MDA-MB-231 cells were transfected with indicated plasmids and kept for 24 hours prior seeding for invasion assay. After 24 hours, 0.5X Three independent experiments were performed in each case to determine statistical significance.

Statistics:
Statistical significance was determined performing Student's two-tailed unpaired t-test as mentioned in the figure legends. GraphPad Prism 6.02 (GraphPad software Inc., La Jolla,CA) was used for the analysis. At least three (n=3) individual experiments were done in each case and data were represented as mean ± SD. p-value < 0.05 were considered to be statistically significant.

CBX4 interacts with hTERT, catalytic component of human telomerase enzyme
CBX4, a well-studied member of polycomb repressive complex 1 (PRC1), plays significant role in DNA damage response and transcriptional regulation through its SUMO E3 ligase activity [14][15][16]32]. Similar to other PcG proteins like BMI1, CBX7 and EZH2 [33], CBX4 shows tumor-promoting effect in several types of cancer [20,21]. We also observed higher shown that oncogenic function of BMI1 intricately depends on the activation of telomerase enzyme activity [10]. The activation of BMI1 was also found to be essential in case of hTERTinduced epithelial to mesenchymal transition (EMT) of oral epithelial cells [12]. Interestingly, CBX4 has been identified as the SUMO E3 ligase of BMI1 and helps to recruit the protein at the DNA damage site [14]. Recently, the role of CBX4 was also observed in the proliferation and metastasis of lung cancer cells through BMI1 regulation [35]. Together these observations led us to examine the role of CBX4 in telomerase activity regulation. As endogenous telomerase activity is higher even in the non-dividing sub-population of HeLa cells [35] and it showed considerable CBX4 expression (Supplemental figure S1A), we selected this cell line for our further biochemical studies. To measure the relative telomerase activity, telomeric repeat amplification protocol (TRAP) was performed either by over expressing or knocking down CBX4 in HeLa cells. Significantly high telomerase activity was found upon CBX4 over expression and the knock-down showed reverse effect ( Figure 1A and 1B). The activity of telomerase enzyme greatly depends on the function of the catalytic component, hTERT which is able to add new hexameric telomere sequences at the end of the chromosome by using hTERC as template. So we subsequently performed co-immunoprecipitation assay to verify whether there is any physical association between CBX4 and hTERT. Indeed we found hTERT interacts robustly with CBX4 ( Figure 1C). Reciprocal co-immunoprecipitation also showed similar association (Supplemental figure S1B). However, we observed additional higher molecular weight bands which were indicative of posttranslational modification of CBX4 and hTERT respectively.
Further we performed co-immunofluorescence experiment after transiently transfecting FLAG-CBX4 in HeLa cells where CBX4 appeared as nuclear foci, a typical characteristic feature of PcG bodies [13] ( Figure 1D). Interestingly, hTERT followed the same distribution pattern and showed prominent co-localization with FLAG-CBX4 as the calculated Pearson's coefficient was greater than 0.5 ( Figure 1D and Supplemental figure S1C, panel I and II). These results clearly indicate towards the involvement of CBX4 in regulating telomerase activity through interaction with catalytic component of human telomerase enzyme, hTERT.

hTERT gets SUMOylated by SUMO 1
The maintenance of telomeric integrity is tightly regulated by multiple factors, including several epigenetic modifications. Along with methylation of sub-telomeric DNA, SUMOylation has also been observed to regulate telomeric length and telomeric integrity at different molecular levels.
Studies in different yeast mutants revealed that accessibility of telomeres to telomerase enzyme also depends on the SUMOylation status of the cells [4]. In addition to that, previous report on phosphorylation and ubiquitination of hTERT itself also showed remarkable effect on the overall activity of telomerase enzyme [36,37]. Given our finding that hTERT interacted with the wellstudied SUMO E3 ligase CBX4 ( Figure 1C), we sought to determine whether hTERT itself is SUMOylated. Indeed through co-immunoprecipitation and reciprocal co-immunoprecipitation, significant interaction was observed between hTERT and SUMO1 ( Figure 2A and Supplemental 13 activity as of vector control cells ( Figure 2D, panel II). HeLa whole cell lysates (WCL) and TRAP lysates were further subjected to immunoblotting to check the expression of FLAG tagged proteins (Supplemental Figure S2A and S2C). So, together these results certainly validate the function of CBX4 as a SUMO E3 ligase for hTERT and also indicate that the catalytic activity of CBX4 is essential for the regulation of telomerase activity.
Regulation of SUMO deconjugation from its substrate has an immense importance in SUMO system and it ensures plasticity of protein interaction network [40]. Six SUMO specific proteases or Sentrin-specific protease (SENPs) are reported so far having role in various disease development [41]. After we found hTERT gets SUMOylated by SUMO E3 ligase CBX4 ( Figure   2D), we wanted to check the effect of most well studied deSUMOylases, SENP1, SENP2 and SENP3 on hTERT SUMOylation. So, HeLa cells were transiently transfected with FLAG-SENP1, SENP2 and SENP3 and interaction was studied between hTERT and SUMO1.
However, substantial reduction in the interaction of hTERT with SUMO1 was observed after over expression of all three SUMO-specific proteases, but the most significant effect was found with SENP3 as compared to SENP1 and SENP2 ( Figure 2E, panel I). Further TRAP assay was performed after over expressing the SENPs in the cells to check the effect of deSUMOylases on telomerase activity. Interestingly, major reduction in telomerase activity was found upon FLAG-SENP3 over expression but SENP1 and SENP2 showed no considerable effect ( Figure 2E, panel II). Protein expression of FLAG-SENP1, SENP2 and SENP3 was confirmed from HeLa whole cell lysates and TRAP-lysates by immunoblotting (Supplemental Figure S2B and S2D). These results indicate that SENP3 acts as the effective deSUMOylase for hTERT and also helps to maintain the activity of the telomerase enzyme.

CBX4 regulates the recruitment of hTERT/ZEB1 complex as a SUMO E3 ligase and represses E-cadherin in breast cancer
Studies in past few years have shown that hTERT has several other functions apart from lengthening of telomeres which are necessary for transformation of normal cells to cancer cells [42]. In case of colorectal cancer, hTERT was found to regulate the expression of E-cadherin, forming complex with an important nuclear transcription factor ZEB1 and showed positive effect in epithelial-to-mesenchymal transition (EMT) [43]. We sought to investigate the role of CBX4  show any significant effect on CDH1 expression in hTERT-depleted condition ( Figure 3B). This result indicates that hTERT is the main molecular player involved in this mechanism. To further elucidate the mechanism of this regulation, occupancy of hTERT was then examined on to the Ebox region of CDH1 (E-cadherin) gene promoter. Interestingly, significant reduction in the enrichment of hTERT was observed on that site through chromatin Immunoprecipitation (ChIP) assay upon FLAG-CBX4 ΔSIM1/2 over-expression ( Figure 3C) as compared to FLAG-CBX4.
Since E-box region of CDH1 has a canonical ZEB1 binding site, we tested for its recruitment at that region and observed differential regulation in the occupancy of the protein upon FLAG-CBX4 and FLAG-CBX4 ΔSIM1/2 over-expression (Supplemental figure S3A). This clearly indicates that the catalytically active wild type CBX4 facilitates the recruitment of hTERT/ZEB1 complex onto CDH1 gene promoter through its SUMO E3 ligase activity. Further, the recruitment of SUMOylated hTERT was also checked on the above promoters through sequencial ChIP and indeed considerable decrease in the enrichment of the complex was observed after FLAG-CBX4 ΔSIM1/2 over-expression ( Figure 3D). These result reconfirmed that the SUMOylation of hTERT is instrumental for the transcriptional repression of CDH1 gene.
Previous reports have already shown the positive effect of hTERT in EMT [47]. We also observed notable reduction in the CDH1 expression upon HA-hTERT over-expression ( Figure   3E). However, the effect was significantly compromised with SUMO-defective mutant of hTERT (HA-hTERT K710R). To further ascertain the importance of hTERT SUMOylation in the repression of CDH1, we over-expressed CBX4 along with wild type hTERT and increasing  Figure 3F). It is evident from the result that CBX4 showed significant repression of CDH1 when co-expressed with wild type hTERT but with hTERT K710R mutant, gradual compromised effect was observed with increasing DNA concentration (1μg, 2 μg, 3 μg) ( Figure 3F). This result suggests that CBX4 effectively inhibits CDH1 gene expression via hTERT SUMOylation.
It was hypothesized that besides acting as SUMO E3 ligase CBX4 also undergoes SUMOylation via an auto-regulatory mechanism [48]. As a member of well-known gene repressive complex PRC1, CBX4 was found to bind the H3K27Me3 mark in a SUMOylation-dependent manner [49]. We observed compromised recruitment of CBX4 itself onto CDH1 promoter upon catalytic  [19,21,23], report suggests that it also suppresses metastasis by interacting with HDAC3 in colorectal carcinoma [22]. Interestingly, by interacting with HDAC1, CBX4 exert oncogenic function by suppressing KLF6 in clear cell renal carcinoma [51]. Therefore, it is quite evident that CBX4 has an ability to interact with different members of various multiprotein complexes which in turn differentially regulates its oncogenic activity. Previously, the effect of Bmi1, another important oncogenic member of PRC1 complex, was observed in the induction of telomerase enzyme activity and immortalization of the human mammary epithelial cells by regulating hTERT promoter [10]. As CBX4 acts as a SUMOE3 ligase for Bmi1, these observations led us to investigate the role of CBX4 itself in the maintenance of telomerase activity and its association to telomerase complex.
Interestingly, through our study we have established a novel interaction between hTERT and SUMO E3 ligase CBX4 which ultimately results in altered telomerase activity ( Figure 1A and B). Previously, the interaction of hTERT and its subsequent effect has been studied with several E3 ubiquitin ligases like MKRN1, CHIP and Plk1 [37,52,53]. As ubiquitination and SUMOylation often work in a coordinated manner we got interested to see whether hTERT undergoes SUMOylation. Indeed we found hTERT gets SUMOylated by SUMO1 (Figure 2A). It is quite well known that for SUMOylation, the target lysine needs to be present within a consensus SUMOylation motif [54]. Through bioinformatics analysis we identified that specific lysine residue at 710 position of RT domain of hTERT and confirmed its involvement by pointmutational approach (Figure 2B and 2C). Overexpression of K710R mutant of hTERT showed reduced hTERT-SUMO1 association as several high-molecular weight (HMW) bands corresponding to SUMOylated hTERT was diminished ( Figure 2C). But, a few HMW bands remained unaltered even in the presence of hTERT K710R mutant, indicating that other potential SUMOylation sites could be involved in this process. Previously mutations in the hTERT promoter have been extensively studied in various cancers [55]. Interestingly, some natural mutations (G682D, P721R, T726M, and K902N) are also found at the RT domain of hTERT in the patients with hematologic disorders and their functional characterization was done [56]. But although two other point mutations (E705Q and V711A) are reported in the TCGA database by the vicinity of our newly identified residue, no detailed analysis has been performed [57,58].
Along with covalent attachment of SUMO to the target lysine residue, the presence of SUMOinteraction motif (SIM) is also necessary for non-covalent SUMO interaction [39,59].
Fascinatingly, SIM can be found in different components of SUMOylation machinery ranging from E1 activating enzyme, members of SUMO E3 ligase family to even in the SUMOsubstrates [60]. CBX4 contains two SIMs which are involved in its SUMO E3 ligase activity as well as mediating non-covalent SUMO-interaction with other proteins [39]. Recently, it has been shown these two SIMs are also responsible for its auto-SUMOylation property [48]. In this current study, we used different SIM deletion mutants of CBX4 to check the effect on hTERT and SUMO1 interaction. Over-expression of SIM1 and SIM2 double mutant of CBX4 (FLAG-CBX4 ΔSIM1/2) showed significant reduction in the hTERT SUMOylation and failed to affect the telomerase activity which confirmed the role of CBX4 as the SUMO E3 ligase of hTERT ( Figure 2D, panel I and panel II). De-SUMOylation plays a major role to maintain the SUMO pool inside the cells [40]. SUMO or Sentrin specific proteases (SENPs) are unique group of isopeptidase enzymes that help not only in the maturation process of SUMO by C-term proteolytic cleavage but also in the deconjugation of SUMO from the substrate protein [41]. So, hTERT, being the catalytic subunit of human telomerase enzyme, prevents shortening of telomere primarily caused due to 'end-replication problem' and catalyzes the addition of telomeric DNA repeats (TTAGGG) at the chromosomal end by using hTERC as a template.
However, recent evidences have suggested the importance of several non-canonical functions of hTERT apart from telomere lengthening, which eventually regulate different hall marks of cancer [61]. Previous report has shown that hTERT promotes epithelial-to-mesenchymal transition (EMT) in gastric cancer [46]. Recently the role of hTERT has also been studied in colorectal cancer through ZEB1 mediated E-cadherin repression and EMT progression [43].
ZEB1 mediated EMT was reported in breast cancer also, where multiple factors are involved [62]. Through our study we found that wild type CBX4 over-expression resulted in ligase of hTERT in this process ( Figure 3D). These findings indicate that SUMOylation through SUMOylation mediated manner as a part of repressive PRC1 complex and helps in the repression of several developmental genes [49]. We observed significant reduction in the occupancy of CBX4 itself and H3K27Me3 at CDH1 gene promoter upon over expression of FLAG-CBX4 ΔSIM1/2 (Supplemental figure S3B and S3C). Furthermore, substantial decrease in the enrichment of H3K27Me3-specific histone methyltransferase EZH2 was also observed at CDH1 gene promoter upon FLAG-CBX4 ΔSIM1/2 over-expression (Supplemental figure S3D) with compared to FLAG-CBX4 which ascertained that the auto-regulation of SUMOylation by CBX4 itself helps in the chromatin recruitment of the protein as a part of repressive chromatin complex.
Induced migratory property and invasiveness of cancer cells were often found as a result of EMT in several types of cancer [50]. We found migration and invasiveness of breast cancer cells majorly depends on SUMOylation of hTERT by SUMO E3 ligase CBX4 ( Figure 5A and B, panel I, II, III and IV). Fascinatingly, both the catalytic mutant of CBX4 and the SUMOdefective mutant of hTERT showed similar compromised effect on migration and invasiveness of breast cancer cells which further re-confirms CBX4 as a SUMO E3 ligase of hTERT ( Figure   4A, 4B, 5A and 5B, panel I, II, III and IV) and strengthens the fact that requirement of hTERT-SUMOylation is essential for EMT progression.
In conclusion, through our present study we have found SUMOylation as a novel posttranslational modification of hTERT and identified polycomb group protein CBX4 as the SUMO E3 ligase for hTERT in this process. Further in depth analysis has revealed the biological significance of hTERT and SUMO1 association in the process of hTERT/ZEB1 mediated EMT in breast cancer cells. Altogether our findings suggest a unique cross talk between repressive   Error bars show standard deviation. Unpaired student's t-test was used to determine p value (*p<0.05, **p<0.010).