The Wnt-responsive transcription factor LEF1 can activate transcription in association with ␤-catenin and repress transcription in association with Groucho. In search of additional regulatory mechanisms of LEF1 function, we identified the protein inhibitor of activated STAT, PIASy, as a novel interaction partner of LEF1. Coexpression of PIASy with LEF1 results in potent repression of LEF1 activity and in covalent modification of LEF1 with SUMO. PIASy markedly stimulates the sumoylation of LEF1 and multiple other proteins in vivo and functions as a SUMO E3 ligase for LEF1 in a reconstituted system in vitro. Moreover, PIASy binds to nuclear matrix-associated DNA sequences and targets LEF1 to nuclear bodies, suggesting that PIASy-mediated subnuclear sequestration accounts for the repression of LEF1 activity.

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after Ϸ22 days, providing an overall reprogramming efficiency of Ϸ0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of Ϸ17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.iPS ͉ reprogramming ͉ OCT4

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3 NPH3 . Under low-intensity BL, CRL3 NPH3 mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi-and polyubiquitinated by CRL3 NPH3 , with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

The ability of the IB␣ protein to sequester dimeric NF-B/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-B/Rel family members. IB␣ can also act in the nucleus as a postinduction repressor of NF-B/Rel proteins. The mechanism by which IB␣ enters the nucleus is not known, as IB␣ lacks a discernible classical nuclear localization sequence (NLS). We now report that nuclear localization of IB␣ is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of IB␣. Furthermore, a region encompassing the second ankyrin repeat of IB␣ is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in IB␣ suggests that cytoplasmic sequestration of the NF-B/Rel-IB␣ complex is a consequence of the mutual masking of the NLS within NF-B/Rel proteins and the import sequence within IB␣. Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), as the ARDs from two other ARD-containing proteins, 53BP2 and GABP␤, are also able to function as nuclear import sequences. We propose that the IB␣ ankyrin repeats define a novel class of cis-acting nuclear import sequences.Directional transport of proteins through the nuclear pore complex provides a powerful regulatory mechanism for controlling gene expression, as illustrated by the NF-B/Rel family of transcription factors (for reviews, see references 3, 5, and 30). Association of the inhibitor of B␣ (IB␣) protein with dimeric NF-B/Rel complexes containing either c-Rel or p65 (RelA) results in the sequestration of the Rel dimer in the cytoplasm, through masking of the nuclear localization sequences (NLSs) within Rel proteins (4,20,26,41,65,77). In response to a variety of extracellular stimuli, including proinflammatory cytokines, viral infection, bacterial lipopolysaccharide, phorbol esters, oxidants, and UV light, IB␣ becomes inducibly phosphorylated at serine residues 32 and 36 (9,10,15,72). The recently identified protein kinase complex, IKK (IB kinase), phosphorylates IB␣ at these N-terminal serine residues and targets IB␣ for ubiquitin-dependent degradation by the 26S proteasome (16,48,60,63,76). Degradation of IB␣ enables the free Rel dimer to translocate to the nucleus and activate B-dependent gene expression. One of the target genes of Rel proteins is the IB␣ gene itself, resulting in the rapid induction of newly synthesized IB␣ protein (1,42,45,70).Several lines of evidence have led to the suggestion that newly synthesized IB␣ can function in the nucleus as a postinduction repressor of B-dependent gene expression. First, ectopically overexpressed IB␣ is readily detected in the nucleus, consistent with the suggestion that IB␣ has a nuclear function (13,50,77). Second, following cytokine stimulation of cells, a significant fraction of newly synthesized endogenous IB␣ appe...

To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O(2) atmosphere (approximately 4% O(2)), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O(2) conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O(2). Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O(2). Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O(2), while a few genes implicated in lineage specification were up-regulated. Although the differences between O(2) conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O(2) favors the molecular mechanisms required for the maintenance of pluripotency.