Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil-forming proteins. Proposed functions for Tpr have included roles as a homo-or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent ␣-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ϳ7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. INTRODUCTIONThe nuclear pore complex (NPC) is a highly complex structure of eightfold rotational symmetry that serves as the gateway for the exchange of cellular material between cytoplasm and nucleus in eukaryotes. Its core structure consists of central globular subunits flanked by a ring-like structure (annulus) at both the NPC's cytoplasmic (outer) and nucleoplasmic (inner) side. Both annuli are attachment sites for fibrils, also arranged in an eightfold symmetrical pattern but of distinctive shape and protein composition (for recent reviews, see Ohno et al., 1998;Stoffler et al., 1999;Ryan and Wente, 2000). Fibrils emanating from the outer annulus exist as short tufts of ϳ50 nm in length, whereas the rectilinear fibrils of 5-7 nm in diameter attached to the inner annulus vary in length. Approximately 60 -100 nm from the NPC's inner annulus, the intranuclear fibrils are laterally interconnected by another ring-like structure, sometimes described as the "terminal ring." Together, terminal ring and fibrils proximal to the NPC proper are considered to represent a structural and functional entity, called the nuclear "fishtrap" or "basket" (Ris, 1989(Ris, , 1991Jarnik and Aebi, 1991;Goldberg and Allen, 1992).Emanating from the terminal ring, additional fibrils project further into the nuclear interior. In amphibian oocytes these fibrils can be seve...

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These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.

BackgroundThe Wiskott–Aldrich syndrome protein (WASp) family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined.MethodsWe performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq) in thymocytes and spleen CD4+ T cells.ResultsWASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF)12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASpL272P and WASpI296T had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus.ConclusionsThese data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0481-6) contains supplementary material, which is available to authorized users.