Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes.

The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box.

Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin ß1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28.

The role of Importin-ßs in the maintenance and lineage commitment of mouse embryonic stem cells.

Members of the Importin-ß family recognize nuclear localization signals (NLS) and nuclear export signals (NES). These proteins play important roles in various nucleocytoplasmic transport processes in cells. Here, we examined the expression patterns of 21 identified Importin-ß genes in mouse embryonic stem cells (mESCs), mouse embryonic fibroblast (MEF) and mESCs differentiated into neural ectoderm (NE) or mesoendoderm (ME). We observed striking differences in the Importin-ß mRNA expression levels within these cell types. We also found that knockdown of selected Importin-ß genes led to suppression of Nanog, and altered the balance of Oct4/Sox2 expression ratio, which is important for NE/ME lineage choice. Furthermore, we demonstrated that knockdown of XPO4, RanBP17, RanBP16, or IPO7 differentially affected the lineage selection of differentiating mESCs. More specifically, knockdown of XPO4 selectively stimulated the mESC differentiation towards definitive endoderm, while concomitantly inhibiting NE differentiation. RanBP17 knockdown also promoted endodermal differentiation with no effect on NE differentiation. RanBP16 knockdown caused differentiation into ME, while IPO7 knockdown inhibited NE differentiation, without obvious effects on the other lineages. Collectively, our results suggest that Importin-ßs play important roles in cell fate determination processes of mESCs, such as in the maintenance of pluripotency or selection of lineage during differentiation.

Eating and rumination behaviour in Brahman grade cattle and crossbred water buffalo fed on high roughage diet.

The purpose of this study is to compare feeding and other behavior and nutrient digestibility of tropical grade Brahman (body weight (BW) = 231 kg ± 12.4; n = 3) and crossbred water buffalo (BW = 300 kg ± 13.9; n = 3). This experiment on digestibility and measures of muscles of mastication utilized one-way, and animal behavior two-way, analysis of variance, respectively. Two video camcorders were installed in each pair of buffalo and Brahman for 24 h period programmed on the 107th, 109th and 111th days of the digestion trials. Frequency and duration of feeding, meal intake, rumination, bolus, chews, drinking, defecating, standing and lying were recorded daily. Muscle diameter of Digastricus, Masseter and Pterygoid and different regions of the tongue were sampled and measured under light microscope using a standard micrometer. Buffalo obtained significantly higher intake of dry matter, roughage, crude protein, total digestible nutrient and metabolized energy than Brahman. This was supported by longer meal duration (P ≤ 0.05), and shorter meal breaks (P ≤ 0.05) of buffalo than Brahman. The diameter of the muscles for mastication was bigger (P ≤ 0.05) in buffalo than in Brahman, which is indicative of stronger chewing ability. Briefly, lesser and slower chewing action; higher intake of roughage and crude protein; and longer resting behavior of crossbred water buffalo than Brahman are all indicative of better digestive and metabolic performance of the buffalo under high roughage feeding conditions.