A cell cycle-dependent co-repressor mediates photoreceptor cell-specific nuclear receptor function.

Photoreceptor cell-specific nuclear receptor (PNR) (NR2E3) acts as a sequence-specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co-repressor, Ret-CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret-CoR identified a multiprotein complex that included E2F/Myb-associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex-related components. Ret-CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret-CoR complex exhibited HDAC activity, co-repressed PNR transrepression function in vitro, and co-repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret-CoR protein was cell-cycle-stage-dependent (from G1 to S). Therefore, Ret-CoR appears to act as a component of an HDAC co-repressor complex that supports PNR repression function in the developing retina, and may represent a co-regulator class that supports transcriptional regulator function via cell-cycle-dependent expression.

Nuclear structure-associated TIF2 recruits glucocorticoid receptor and its target DNA.

Assembly of multi-protein complexes on promoter and enhancer elements is a prerequisite for onset of gene transcription. At the beginning of this process, transcription factors are thought to act as nucleating centers for complex formation through the binding of their target DNA sequences, and thereafter recruit coactivators. Here, we investigated this process of assembly by determining the distribution of the glucocorticoid receptor (GR) and its coactivator, TIF2. Both endogenously and ectopically expressed TIF2 were shown to form foci in the nucleus, and GR could be recruited to the TIF2 foci upon GR agonist but not antagonist treatment. Moreover, we show that the coactivators, p300 and PCAF, are also recruited to the TIF2 foci. The TIF2 foci could recruit GR carrying a microinjected GR responsive element. We propose that TIF2 provides a nuclear compartment that allows the assembly of multi-protein complexes required for GR-mediated gene activation.

Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors which belong to the nuclear receptor family. We examined whether PPARalpha agonists and resveratrol, a polyphenol contained in grapes, protect the brain against ischemia. To investigate whether resveratrol activates PPARs, we performed a cell-based transfection activity assay using luciferase reporter plasmid. PPARalpha and PPARgamma were activated by resveratrol in primary cortical cultures and vascular endothelial cells. Resveratrol (20 mg/kg, 3 days) reduced infarct volume by 36% at 24 h after middle cerebral artery occlusion in wild-type mice. The PPARalpha agonists fenofibrate (30 mg/kg, 3 days) and Wy-14643 (30 mg/kg, days) exerted similar brain protection. However, resveratrol and fenofibrate failed to protect the brain in PPARalpha knockout mice. The data indicate that PPARalpha agonists protect the brain through PPARalpha.

LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.

The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERalpha (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORalpha (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions -2, +2, +3, and +6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.

When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?

During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.