Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t 1/2 ] of 4 to 6 min) of agonist-and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G 0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin ␣. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t 1/2 ‫؍‬ 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin ␣. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1 ؊ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.The predominant pathway for the nuclear import of transcription factors and other nuclear regulatory proteins originates with the interaction of importin ␣-like proteins (also called karyopherin ␣, Rch1/hSRP␣, hSRP1/NPI-1, and pendulin/OHO31) with specific nuclear localization sequences (NLSs), which contain closely spaced arrangements of five to eight basic amino acids (31,62,64). For DNA sequence-specific transcription factors, NLSs generally colocalize with their DNA binding domains (DBDs), which appears to reflect a coevolutionary selective pressure to ensure that proteins that bind DNA are able to access the nucleus (52). Nuclear export, by contrast, occurs through alternative pathways, which for many proteins involves the binding of CRM1 (exportin 1) to hydrophobic nuclear export sequences (26,90).However, some transcription factors, including the glucocorticoid hormone receptor (GR), contain additional NLSs that occur in other regions of the proteins (69,89,95,99). In at least some instances, the presence of these additional NLSs has been found to reflect a requirement for specialized or tightly regulated nuclear localization of the protein. For example, the nuclear localization potential of one of the two NLSs in the adenovirus E1A protein is active only during early development (92), while two of the three c-abl NLSs promote nuclear localization of c-abl only in certain cell types (97, 99). T...

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hoxindependent and -dependent pathways, respectively. DEVELOPMENT 4100 system to derive a conditional null allele. In agreement with previous studies (Chawengsaksophak et al., 2004), loss of Cdx2 in the postimplantation embryo resulted in axial truncation posterior to the forelimb. This axial truncation, together with the nature of many of the genes impacted by Cdx2 loss, suggested that precocious cessation of the generation of presomitic mesoderm (PSM) is the primary basis for this phenotype. To further define the role of Cdx2 and to elucidate the mechanistic basis for this phenotype, we sought to determine whether any of the affected genes were direct Cdx2 targets. Chromatin immunoprecipitation (ChIP) demonstrated occupancy of the Wnt3a, Cyp26a1 and T promoters by Cdx2 in vivo. Moreover, all of these promoters harbor functional Cdx response elements (CDREs) as determined by transfection analysis or transgenic assays. Thus, Cdx2 directly regulates the expression of multiple players essential for the development of the posterior embryo. Taken together with previous work, these findings suggest that Cdx2 is required to couple the generation of paraxial mesoderm through multiple Hox-independent mechanisms with Hoxdependent AP vertebral patterning. MATERIALS AND METHODS Gene targeting and the generation of Cdx2 -/-mutantsA 5 kb fragment of genomic Cdx2 sequence encompassing the first intron through to the 3Ј UTR was subcloned into pBluescript II KS + . A floxed thymidine kinase/neomycin resistance cassette (loxPGK-TK-Neolox) (Iulianella and Lohnes, 2002) was cloned into the BglII site in intron 1, and a single loxP site was inserted into the NruI site in intron 2, generating the targeting vector (see Fig. S1A in the supplementary material). R1 embryonic stem cells were electroporated with...

Cdx1, Cdx2 and Cdx4 encode homeodomain transcription factors that are involved in vertebral anterior-posterior (AP) patterning. Cdx1 and Cdx2 are also expressed in the intestinal epithelium during development, suggesting a role in this tissue. Intestinal defects have not been reported in Cdx1 null mutants, while Cdx2 null mutants die at embryonic day 3.5 (E3.5), thus precluding assessment of the null phenotype at later stages. To circumvent this latter shortcoming, we have used a conditional Cre-lox strategy to inactivate Cdx2 in the intestinal epithelium. Using this approach, we found that ablation of Cdx2 at E13.5 led to a transformation of the small intestine to a pyloric stomach-like identity, although the molecular nature of the underlying mesenchyme remained unchanged. Further analysis of Cdx1-Cdx2 double mutants suggests that Cdx1 does not play a critical role in the development of the small intestine, at least after E13.5.

Steroid hormone receptors act to regulate specific gene transcription primarily as steroid-specific dimers bound to palindromic DNA response elements. DNA-dependent dimerization contacts mediated between the receptor DNA binding domains stabilize DNA binding. Additionally, some steroid receptors dimerize prior to their arrival on DNA through interactions mediated through the receptor ligand binding domain. In this report, we describe the steroid-induced homomeric interaction of the rat glucocorticoid receptor (GR) in solution in vivo. Our results demonstrate that GR interacts in solution at least as a dimer, and we have delimited this interaction to a novel interface within the hinge region of GR that appears to be both necessary and sufficient for direct binding. Strikingly, we also demonstrate an interaction between GR and the mineralocorticoid receptor in solution in vivo that is dependent on the ligand binding domain of GR alone and is separable from homodimerization of the glucocorticoid receptor. These results indicate that functional interactions between the glucocorticoid and mineralocorticoid receptors in activating specific gene transcription are probably more complex than has been previously appreciated.The effects of corticosteroids are determined through asymmetric distribution of the mineralocorticoid and glucocorticoid nuclear hormone receptors (MR and GR) and the protective effects of 11␤-hydroxysteroid dehydrogenase, which selectively metabolizes glucocorticoids (2, 20, 31). MR is highly sensitive to both mineralocorticoids and glucocorticoids, while GR responds only to higher levels of glucocorticoids and is mostly insensitive to mineralocorticoids.Coordinate signaling by GR and MR is specifically relevant to tissues such as the brain, where an abundance of MR and GR in areas such as the hippocampus is accompanied by an absence of 11␤-hydroxysteroid dehydrogenase (14). Indeed, the effects of GR and MR are critical for homeostatic control of CAl pyramidal neurons, where the two receptors differentially mediate the control of ion regulation and transmitter responsiveness (27). Thus, MR and GR signaling influence memory, mood, and neuronal survival. Elevated cortisol levels correlate with depression and other stress-related psychopathologies and with a long-term attenuation of serotonin signaling (28,29,61).GR and MR function predominantly to regulate specific gene expression patterns through palindromic response elements that accommodate receptor dimers (1). The DNA binding domains (DBDs) of the steroid hormone receptors are highly conserved. As a result, GR and MR, as well as progesterone receptors (PR) and androgen receptor (AR), bind in closely related ways to broadly overlapping response elements. Homodimerization contacts mediated through the receptor DBDs occur on DNA binding and are mediated through specific contacts involving residues in the second zinc finger of the receptor DBDs (38).The potential for transcriptional regulation via heteromeric complexes of these steroid receptors has re...