Jeong-Hoon Joo

Nuclear Speckle-Associated Protein Pnn/DRS Binds to the Transcriptional Corepressor CtBP and Relieves CtBP- Mediated Repression of the E-Cadherin Gene

ABSTRACT 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.

Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis

Pinin (Pnn), a nuclear speckle-associated protein, has been shown to function in maintenance of epithelial integrity through altering expression of several key adhesion molecules. Here we demonstrate that Pnn plays a crucial role in small intestinal development by influencing expression of an intestinal homeobox gene, Cdx2. Conditional inactivation of Pnn within intestinal epithelia resulted in significant downregulation of a caudal type homeobox gene, Cdx2, leading to obvious villus dysmorphogenesis and severely disrupted epithelial differentiation. Additionally, in Pnn-deficient small intestine, we observed upregulated Tcf/Lef reporter activity, as well as misregulated expression/distribution of beta-catenin and Tcf4. Since regulation of Cdx gene expression has been closely linked to Wnt/beta-catenin signaling activity, we explored the possibility of Pnns interaction with beta-catenin, a major effector of the canonical Wnt signaling pathway. Co-immunoprecipitation assays revealed that Pnn, together with its interaction partner CtBP2, a transcriptional co-repressor, was in a complex with beta-catenin. Moreover, both of these proteins were found to be recruited to the proximal promoter area of Cdx2. Taken together, our results suggest that Pnn is essential for tight regulation of Wnt signaling and Cdx2 expression during small intestinal 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.

Role of Pinin in neural crest, dorsal dermis, and axial skeleton development and its involvement in the regulation of Tcf/Lef activity in mice.

Previous in vitro studies have indicated multiple and varied roles of Pinin (PNN); however, its in vivo role has remained unclear. Here, we report generation of null, hypomorphic, and conditional Pnn alleles in mice. We found that insertion of neomycin‐resistance cassette into intron 8 of Pnn resulted in knockdown of Pnn, which allowed Pnn hypomorphic embryos to pass peri‐implantation lethality. These mice are lethal at perinatal stages and exhibit defects in the cardiac outflow tract, palate, dorsal dermis, and axial skeleton. Since Wnt/β‐catenin signaling has been shown to play pivotal roles in development of all tissues affected by Pnn hypomorphism, we speculated that Pnn may affect Wnt/β‐catenin signaling. Supporting this view, we demonstrate abnormal activities of Tcf/Lef transcription factors, and alterations in β‐catenin level in multiple Pnn hypomorphic tissues. Taken together, the data suggest that Pnn plays important roles during mouse development through its involvement in regulation of Tcf/Lef activity. Developmental Dynamics 236:2147–2158, 2007.

Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells

PURPOSE We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context. METHODS Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis. RESULTS Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement. CONCLUSIONS Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.

Corepressor CtBP and Nuclear Speckle Protein Pnn/DRS Differentially Modulate Transcription and Splicing of the E-Cadherin Gene

ABSTRACT 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.