Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium.

PURPOSE: GG-H whole transcriptome array analysis suggested involvement of PININ (PNN) in the alternative splicing of multiple long non-coding RNAs (lncRNAs). To further investigate PNN's role in regulating the alternative splicing of lncRNAs in a corneal epithelial context, we performed detailed analyses for detecting and identifying alternatively spliced lncRNAs. METHODS: Total RNA was isolated from PNN knockdown human corneal epithelial (HCET) cells or Pnn-deficient mouse corneas, and subjected to real-time-PCR (RT-PCR) assays, and the alternatively spliced lncRNAs were counted. Alternatively spliced lncRNAs were detected with in situ hybridization with variant-specific RNA probes on human cornea sections. RESULTS: Our analysis uncovered PNN's impact on the transcript levels of several lncRNAs including Linc00085 and HAS2-AS1. Interestingly, a mouse ortholog of HAS2-AS1, Has2as, clearly exhibited a differential splicing pattern among three major splice variants in the Pnn-deficient mouse cornea. The sequence analyses and quantification of splice variants of candidate lncRNAs, including RP11-295B20.2, RP11-18I14.1, and RP11-322M19.1, demonstrated complex configuration of their splicing changes, with a significant impact of PNN on the process. Knockdown of PNN in HCET cells led to specific changes in the inclusion of multiple cassette exons as well as in the use of alternative splice sites in RP11-322M19.1 and RP11-18I14.1, resulting in considerable net changes in the ratio between the splice variants. Finally, in situ hybridization analyses revealed the presence of RP11-295G20.2 in the nuclei of corneal epithelial cells, but not in the stromal cells of the human cornea, while RP11-322M19.1 was present in epithelial and non-epithelial cells. CONCLUSIONS: The data suggest PNN's role in the alternative splicing of a specific subset of lncRNAs might have a significant impact on the corneal epithelium.

Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development.

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

Disruption of mouse corneal epithelial differentiation by conditional inactivation of pnn.

Purpose. To investigate the specific role of Pinin (Pnn) in the development of anterior eye segment in mice. Methods. Conditional inactivation of Pnn in the developing surface eye ectoderm and lens was achieved by creating mice carrying a Pnn null and a floxed Pnn allele as well as a Pax6-Cre-GFP (Le-Cre) transgene. The resultant Pnn conditional knockout mice were examined by histologic and immunohistologic approaches. Results. Pax6-Cre-mediated deletion of Pnn resulted in severe malformation of lens placode-derived tissues including cornea and lens. Pnn mutant corneal epithelium displayed the loss of corneal epithelial identity and appeared epidermis-like, downregulating corneal keratins (K12) and ectopically expressing epidermal keratins (K10 and K14). This squamous metaplasia of Pnn mutant corneal epithelium closely correlated with significantly elevated beta-catenin activity and Tcf4 level. In addition, Pnn inactivation also led to misregulated level of p68 RNA helicase in mutant corneal epithelium. Conclusions. These data indicate that Pnn plays an essential role in modulating and/or orchestrating the activities of major developmental factors of anterior eye segments.

Corepressor CtBP and nuclear speckle protein Pnn/DRS differentially modulate transcription and splicing of the E-cadherin gene.

CtBP is a transcriptional corepressor with tumorigenic potential that targets the promoter of the tumor suppressor gene E-cadherin. Pnn/DRS (Pnn) is a "nuclear speckle"-associated protein involved in mRNA processing as well as transcriptional regulation of E-cadherin via its binding to CtBP. Here, we show that CtBP can recruit Pnn to CtBP-associated complexes, resulting in Pnn-dependent chromatin remodeling at the E-cadherin promoter. In addition, CtBP and Pnn can differentially modulate E-cadherin mRNA splicing, with polymerase II serving as an interface in this event. Therefore, the Pnn/CtBP functional interplay represents a novel mechanism linking the corepressor CtBP and Pnn to the transcription-coupled mRNA splicing of a major tumor suppressor gene. Our findings implicate the existence of the molecular switches involved in tumorigenesis, which coordinate promoter-specific events and mRNA processing, by serving as bridging elements between the regulatory complexes both at gene promoters and within the mRNA splicing machineries.

Nuclear speckle-associated protein Pnn/DRS binds to the transcriptional corepressor CtBP and relieves CtBP-mediated repression of the E-cadherin gene.

Previously, we have shown that pinin/DRS (Pnn), a 140-kDa nuclear and cell adhesion-related phosphoprotein, is involved in the regulation of cell adhesion and modulation of the activity of multiple tumor suppressor genes. In the nucleus Pnn is concentrated in the "nuclear speckles," zones of accumulation of transcriptional and mRNA splicing factors, where Pnn is involved in mRNA processing. Alternatively, other roles of Pnn in gene regulation have not yet been established. By utilizing in vitro pull-down assays, in vivo interaction studies, and immunofluorescence in combination with overexpression and RNA interference experiments, we present evidence that Pnn interacts with the known transcriptional corepressor CtBP1. As a consequence of this interaction Pnn was capable of relieving the CtBP1-mediated repression of E-cadherin promoter activity. Our results suggest that the interaction of Pnn with the corepressor CtBP1 may modulate repression of transcription by CtBP1. This interaction may reflect the existence of coupling factors involved in CtBP-mediated transcriptional regulation and mRNA processing events.