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We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT.We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3epsilon, 14-3-3tau, 14-3-3zeta and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3sigma and Snail1 expression in human colorectal tumors.We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.

Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.