Multi-dimensional Proteomics Reveals A Role Of UHRF2 In The Regulation Of Epithelial-Mesenchymal Transition(EMT)

UHRF(ubiquitin-like, containing PHD and RING finger domains, inverted CCAAT Box-Binding Protein of 90 KDa) family contains four members characterized with multiple domains in structure. The founding member of this family, UHRF1 is the best characterized. UHRF1 has been reported to play critical roles in various processes, including the maintenance of DNMT1-mediated DNA methylation. UHRF2 has similar sequence and domain architectures as UHRF1. This similarity suggests a potential functional conservation between these two proteins. Like UHRF1, UHRF2 also recognizes hemimethylated DNA substrates and H3K9me2/3, and interacts with DNMTs and histone methyltransferase G9 a in vitro. However, there are substantial differences between UHRF1 and UHRF2. UHRF2 cannot rescue the DNA methylation defect in Uhrf1-/- ES cells due to its inability to recruit DNMT1 to replication foci during cell cycle S phase. In addition, UHRF2 was reported as a specific binder of 5hm C with the structure of UHRF2-SRA domain having a preferential binding of 5hm C while UHRF1 does not have this binding preference. Like UHRF1, UHRF2 was also implicated in tumors. But reports about the role of UHRF2 in tumors are contradictory and uncertain. Some studies demonstrated that UHRF2 behaves like a tumor suppressor to inhibit the inappropriate cell cycle progression, whereas other studies suggested potential oncogenic characteristics of UHRF2 with upregulated expression in cancers. Because of the limited number of studies on the involvement of UHRF2 in tumorigenesis, the precise biological function of UHRF2 in cancer and whether it also functions like UHRF1 remain to be investigated.Metastasis is an important characteristics of cancer and responsible for more than 90% of cancer associated mortality. Metastasis of cancer cells is a complex process that is partly regulated by activation of epithelial to mesenchymal transition(EMT) to acquire the ability to invade and metastasize. During EMT, epithelial cells lose cell–cell contacts and cell polarity, and acquire mesenchymal-like characteristics with increased ability of migration and invasion. EMT is orchestrated by transcription factor cascades that regulate the expression of proteins involved in cell–cell contacts, cell polarity, cytoskeleton structure and extracellular matrix degradation. Researches about EMT increased in recent years, but the mechanism about how EMT-TFs function need further investigation.Mass spectrometry(MS)-based proteomics is a powerful approach for large scale protein analysis in biological research. Our lab has developed a fast, label-free quantification workflow(Fast-quan) for protein identification, in which 7,000 proteins can be identified and quantified with 12 hrs of MS running time. This has enabled analysis of multiple samples. We also developed a concatenated tandem array of transcription factor response elements(cat TFRE) pull-down assay that allows for enrichment and identification of endogenous transcription factors(TFs). The combination of measuring changes in DNA binding activity of TFs and proteome-wide profiling of protein abundance allows us to correlate TF activity with target genes in response to exogenous stimulation. Thus, the proteome-wide identification of activated TFs when cells are perturbed can provide important biological clues about the mechanisms and signal transduction pathways.To explore the functions of UHRF2, we used multiple proteomics tools including whole proteome profiling, cat TFRE pull-down-MS, IP-MS and Ch IP-seq to investigate what UHRF2 does to the proteome when over-expressed in a cancer cell line. We profiled the proteomes upon UHRF2 overexpression in a gastric cancer cell line MKN74 from two biological and two operational replicates. A total of 7,662 unique proteins were identified with the detection of at least 1 unique peptide at 1%FDR. One hundred ninety-two(192) proteins(2.5% of the proteome) appeared to be significantly increased in their abundances upon UHRF2 overexpression(> 1.5-fold change, p-value < 0.05). Similarly, 297 proteins(3.8% of the proteome) were considered as decreased. Notably, in GO terms, increased proteins are mainly annotated as residing in nucleus, macromolecular complex and cytoplasm while decreased proteins are mainly annotated as residing in cell-cell junction, basal lamina and endomembrane system. It demonstrates that UHRF2 represses a number of epithelial markers including CDH1, JUP, TJP1, DSG2, INADL, SPINT2, CXADR, SPINT1 and TJP2 and stabilizes some important nuclear proteins including p53.Overall, altered expression of proteins by UHRF2 overexpression suggests an association of UHRF2 with cancers and metastasis.To gain further insights into the driving force for altered proteins expression upon UHRF2 overexpression, we used cat TFRE to measure TF-DNA binding activity. This allowed us to measure a group of low abundance transcription factors that were not covered in the previous profiling experiments. We detected 581 TFs and 537 coregulators(Co Rs) out of six replicates with unique peptides(us Pepts, 1% FDR at peptide level). Seventeen(17) TFs and 8 Co Rs were upregulated upon UHRF2 overexpression as compared with controls. Notably, five TFs are annotated as EMT-TFs according to the previous literature reports. In summary, The TF DNA binding activity data and protein profiling data correlate well and both support a model in which overexpression of UHRF2 leads to the activation of EMT-TFs and an altered expression of EMT proteins.To provide further evidence for the involvement of UHRF2 in EMT process, we depleted UHRF2 in aggressive cell lines SGC7901 and BGC823 using CRISPR-Cas9 system with two different guide RNAs(g RNAs). We used the transwell assay to evaluate the cell migration ability. Compared to the control group, UHRF2 knockout with either g RNA1 or g RNA2 caused a significant decrease in cell migration in different clones of SGC7901 cells. The reduction in migration ability upon UHRF2 knockout was also observed in another GC cell lines BGC823. Furthermore, we determined the invasion ability of cells using transwell assay with Matrigel on the insert surface. We found that the silencing of UHRF2 significantly suppressed the invasion of SGC7901. We found that loss of UHRF2 caused a profound morphological change for SGC7901 cells from mesenchymal-like morphology to epithelial morphology and induced the formation of Spheroid. these data suggest that UHRF2 plays a causal role in cell motility.We performed chromatin immunoprecipitation(Ch IP) experiments with an UHRF2 antibody using Ig G as a control. The results showed a significant association of UHRF2 with CDH1 promoter which was not detected in Ig G control group. Taken together, we show that UHRF2 physically associated with the promoter region of CDH1 and may therefore function as a transcriptional co-regulator for its expression. To gain more mechanistic insights into the action of UHRF2 in regulating the EMT process, we carried out IP-MS experiments to discover proteins that physically associate with UHRF2. The IP-MS data revealed physical interactions of UHRF2 with several epigenetic regulation complexes, consistent with its known biological function; furthermore, the identification of TCF7L2, a TF that regulates EMT, provides additional evidence for the involvement of UHRF2 in EMT processes.In summary, we provided evidence to demonstrate a role of UHRF2 in cell motility and invasion through the regulation of the Epithelial-Mesenchymal Transition(EMT) process by acting as a transcriptional co-regulator of the EMT-transcription factors(TFs). We ectopically expressed UHRF2 in gastric cancer cell lines and performed multi-dimensional proteomics analyses. The proteome profiling suggested the repression of cell-cell adhesion by UHRF2; the analysis of proteome-wide TF DNA binding activities revealed the up-regulation of many EMT-TFs. These data suggested that UHRF2 is a regulator of cell motility and the EMT program. Indeed, cell invasion experiments demonstrated that silencing of UHRF2 in aggressive cells impaired their abilities of migration and invasion in vitro. Further Ch IP-seq suggested that UHRF2 genomic binding motifs coincide with several TF binding motifs including EMT-TFs, and the binding of UHRF2 to CDH1 promoter was validated by Ch IP-q PCR. Moreover, the interactome analysis with IP-MS uncovered the interaction of UHRF2 with TFs including TCF7L2 and several protein complexes that regulate chromatin remodeling and histone modifications, suggesting that UHRF2 is a transcription co-regulator together with TFs like TCF7L2 to regulate the EMT process. Our study identified a role of UHRF2 in EMT and tumor metastasis and demonstrated an effective approach to obtain clues of UHRF2 function without prior knowledge through combining evidence from multi-dimensional proteomics analyses.