Nuclear proteins are selectively imported into the nucleus by transport factors such as importin-alpha and importin-beta. Here, we show that the expression of importin-alpha subtypes is strictly regulated during neural differentiation of mouse embryonic stem (ES) cells, and that the switching of importin-alpha subtype expression is critical for neural differentiation. Moreover, reproducing the switching of importin-alpha subtype expression in undifferentiated ES cells induced neural differentiation in the presence of leukaemia inhibitory factor (LIF) and serum, coordinated with the regulated expression of Oct3/4, Brn2 and SOX2, which are involved in ES-neural identity determination. These transcription factors were selectively imported into the nucleus by specific subtypes of importin-alpha. Thus, importin-alpha subtype switching has a major impact on cell differentiation through the regulated nuclear import of a specific set of transcription factors. This is the first study to propose that transport factors should be considered as major players in cell-fate determination.

Elevated oxidative stress has been suggested to be associated with the features of Down’s syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation‐derived products, 13‐hydroperoxy‐9Z,11E‐octadecadienoic acid and 4‐hydroxy‐2‐nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation‐sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13‐hydroperoxy‐9Z,11E‐octadecadienoic acid and/or 4‐hydroxy‐2‐nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation.However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain)-in the N-terminal region of importin α within the conventional importin β binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.

Transport of functional molecules across the nuclear membrane of a eukaryotic cell is regulated by a dedicated set of transporter proteins that carry molecules into the nucleus or out of the nucleus to the cytoplasm for homeostasis of the cell. One of the categories of cargo molecules these transporters carry are the molecules for cell cycle regulation. Therefore, their role is critical in terms of cancer development. Any misregulation of the transport factors would means aberrant abundance of cell cycle regulators and might have consequences in cell cycle progression. While earlier studies have focussed on individual transport related molecules, a collective overview of how these molecules may be dysregulated in breast cancer is lacking.Using genomic and transcriptomic datasets from TCGA (The Cancer Genome Atlas) and microarray platforms, we carried out bioinformatic analysis and provide a genetic and molecular profile of all the molecules directly related to nucleocytoplasmic shuttling of proteins and RNAs. Interestingly, we identified that many of these molecules are either mutated or have dysregulated expression in breast cancer. Strikingly, some of the molecules, namely, KPNA2, KPNA3, KPNA5, IPO8, TNPO1, XPOT, XPO7 and CSE1L were correlated with poor patient survival. This study provides a comprehensive genetic and molecular landscape of nucleocytoplasmic factors in breast cancer and points to the important roles of various nucleocytoplasmic factors in cancer progression. This data might have implications in prognosis and therapeutic targeting in breast cancer.

AbstractThe nuclear transport of proteins is important for facilitating appropriate nuclear functions. The proteins of the importin α family play key roles in nuclear transport as transport receptors for a huge number of nuclear proteins. Additionally, these proteins are also reported to possess other functions, including chromatin association and gene regulation. However, these non-transport functions of importin α are not yet fully understood, especially its molecular interaction with the chromatin and its consequences. Here, we report novel molecular characteristics of importin α involving the binding to multiple regions of chromatin. We newly identified a DNA binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain) in the N terminal region of importin α within the conventional importin β binding (IBB) domain which is necessary for the nuclear transport of cargo proteins. We propose a “stroll and locate” model to explain how importin α associates with double-stranded DNA. This is the first study to delineate interaction between importin α with the chromatin via the NAAT domain, indicating the bifunctionality of the importin α N terminal region for the nuclear transport and the chromatin association.