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.We studied a molecule, pinin (Pnn/DRS/memA), a 140-kDa phosphoprotein associated with the desmosome and localized in the nucleus of various tissues and cultured cell lines (4, 40-42, 52, 53, 62), which seems to play a key role in the establishment and maintenance of epithelia (53, 56). The expression of exogenous Pnn in transformed cells dramatically altered the recipient cells' morphology, driving them to a more epithelial phenotype. Expression of Pnn was linked to the expression of genes such as E-cadherin, p21 cip/waf , MIC-1, and Rho-A, which impact epithelial adhesion, proliferation, and cell motility (55). These observations suggest that Pnn may play an integral role in epithelial-cell-specific gene expression.Transfection experiments revealed that the majority of expressed Pnn is found within the nucleus, exhibiting a diffuse nucleoplasmic and nuclear speckle distribution. However, little, if any, exogenous Pnn was localized to cell-cell adhesion sites. This raises the possibility that Pnn may be exerting its effect predominantly via interaction with nuclear components, perhaps involving mRNA transcription and processing (48,62). The proposed functions of nuclear speckles include a role as storage compartments for molecular components involved in gene transcription, such as a subpopulation of polymerase II, and mRNA processing machinery, such as the SR family of proteins. SR proteins distinguished by their serine/argininerich motifs are required for constitutive mRNA splicing and regulation of alternative splice site selection (6,30,36,37,47,58). Previous two-hybrid and colocalization experiments revealed that Pnn indeed binds to, and colocalizes with, the SR protein family members SRp75 and SRm300, known components of the spliceosome machinery, and a novel SR protein (9,47,62,65). In addition, Pnn was demonstrated to contribute to the alternative splice site selection in splicing reporter assays, sp...

The results suggest that Pnn and SR-rich proteins may be part of a multiprotein complex within the nucleus and may be involved in pre-mRNA processing.

Our data suggest that in vivo CSLO imaging of EYFP-RGC expression and SD-OCT measured NFL thickness are fast and reliable methods that longitudinally track neurodegenerative progression following ONC injury. Neurodegenerative changes in NFL thickness measured by SD-OCT imaging have the same overall trajectory as those observed by CSLO for RCD; however, changes in NFL thickness initially lag behind in vivo RGC soma counts with a slower decline in overall measurable change.

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/␤-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/␤-catenin signaling. Supporting this view, we demonstrate abnormal activities of Tcf/Lef transcription factors, and alterations in ␤-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.

The strong correlation between the combined layer thickness and histologic cell counts validates manual OCT segmentation as a method of monitoring cell loss in the RGCL. A retinal thickness map assessed if combined NFL and IPL thickness loss in Brn3b(-/-) eyes was topographically specific. Generalized RGC and combined NFL and IPL loss was observed in the Brn3b(-/-) retinas, in contrast to topographically specific RGC loss observed in glaucomatous DBA2/J eyes.

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 β-catenin and Tcf4. Since regulation of Cdx gene expression has been closely linked to Wnt/β-catenin signaling activity, we explored the possibility of Pnn’s interaction with β-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 β-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.

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.

Purpose Recent advances in technology now provide tools capable of tracking genome-wide expression changes occurring in progressive pathological processes. The present experiments were carried out to determine if acetylcholine receptor α 6 subunit (Chrna6) is a reliable retinal ganglion cell (RGC) marker in adult mouse eyes and if Chrna6 expression can be used to track progressive loss of RGCs, such as is observed in the DBA/2J glaucoma model. Methods Data sets derived from the BXD strains were used to extract gene expression signatures for RGCs. Pooled retinas from DBA/2J or C57BL/6J cases at 1–3 months, 12 months, and 16–17 months were prepared for gene-array and RT-PCR analysis. Globes were fixed in paraformaldehyde and sectioned for immunofluorescence with antibodies against Chrna6. Results Chrna6 has a cellular expression signature for RGCs with high correlation to Thy1 (r = 0.65), a recognized RGC marker. Immunofluorescence experiments confirm that in the young and adult mouse retina, Chrna6 is preferentially expressed by RGCs. We further show that C3H/HeJ retinas, which lack photoreceptors, also express Chrna6 in the RGC layer. Gene expression array analyses, confirmed by RT-PCR, show progressive loss of Chrna6 expression in retinas of the DBA/2J glaucomatous mouse retinas. Conclusions Quantitative trait locus analysis provides support for Chrna6 as a RGC marker. Chrna6 expression decreases with death of RGCs in glaucomatous DBA/2J mice and after optic nerve crush injury, further supporting Chrna6 as a reliable RGC marker. High expression of RGC Chrna6 in the absence of photoreceptors is suggestive that Chrna6 expression by RGCs is independent of photoreceptor-derived stimuli.