Biomedical subjects

Gordon L Hager

Publications and source records attributed to Gordon L Hager.

33 records · Page 2Linked to original sources

PubMed2004

Molecular chaperones function as steroid receptor nuclear mobility factors.

Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and/or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.

Active Transport, Cell Nucleus↗

PubMed2004

Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage.

The p53 tumor suppressor protein plays a critical role in mediating cellular response to stress. Upon DNA damage, post-translational modifications stabilize and activate this nuclear phosphoprotein. To determine the effect of phosphorylation site mutants in the context of the whole p53 protein, we performed reporter assays in p53 and MDM2 knockout mouse embryonic fibroblasts transfected with full-length p53 constructs. We show that mutation of S37 causes a decrease in p53 transcriptional activity compared to wild-type p53. Our data further suggest that the dephosphorylation of p53 at S37 is a regulated event involving protein phosphatase 2A (PP2A). Coimmunoprecipitation and immunofluorescence microscopy studies demonstrate that PP2A and p53 associate with one another in vivo following gamma-irradiation. Consistent with these observations, phosphorylated S37 accumulates in cell extracts prepared from gamma-irradiated Molt-4 cells in the presence of okadaic acid. Furthermore, in vitro phosphatase assays show that PP2A dephosphorylates p53 at S37. These results suggest that dephosphorylation of p53 at S37 plays a role in the transcriptional regulation of the p53 protein in response to DNA damage.

Catalytic Domain↗

PubMed2004

Rapid glucocorticoid receptor exchange at a promoter is coupled to transcription and regulated by chaperones and proteasomes.

Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.

Animals↗

PubMed2004

Subnuclear trafficking and gene targeting by steroid receptors.

Through the use of novel imaging techniques, we have observed direct steroid receptor binding to a tandem array of a hormone-responsive promoter in living cells. We found that the glucocorticoid receptor (GR) exchanges rapidly with regulatory elements in the continued presence of ligand. We have also reconstituted a GR-dependent nucleoprotein transition with chromatin assembled on promoter DNA, and we discovered that GR is actively displaced from the chromatin template during the chromatin remodeling process. Using high-intensity UV laser crosslinking, we have observed highly periodic interactions of GR with promoter chromatin. These periodic binding events are dependent on GR-directed hSWI/SNF remodeling of the template and require the presence of ATP. Both the in vitro and in vivo results are consistent with a dynamic model ("hit-and-run") in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is simultaneously lost from the template. We also find that receptor mobility in the nucleoplasm is strongly enhanced by molecular chaperones. These observations indicate that multiple mechanisms are involved in transient receptor interactions with nucleoplasmic targets.

Animals↗

PubMed2003

Formation of higher-order secondary and tertiary chromatin structures by genomic mouse mammary tumor virus promoters.

Agarose multigel electrophoresis has been used to characterize the structural features of isolated genomic mouse mammary tumor virus (MMTV) promoters. The mouse 3134 cells used for these studies contain approximately 200 stably integrated tandem repeats of a 2.4-kb MMTV promoter fragment. Inactive, basally active, and hormonally activated genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in low-salt nuclear extracts, and electrophoresed in multigels consisting of nine individual agarose running gels. Specific bands were detected and characterized by Southern and Western blotting. We find that transcriptionally inactive promoters contain TBP and high levels of histone H1, and are present to varying extents in both untreated and dexamethasone (DEX)-treated 3134 cells. In contrast, the basally active promoter, present in untreated cells, is bound to RNA Pol II, TBP, and Oct1, contains acetylated H3 tail domains, and is depleted of histone H1. The DEX-activated promoter possessed similar composition as the basal promoter, but also contains stably bound Brg1. Strikingly, all forms of the MMTV promoter condense into higher-order secondary and/or tertiary chromatin structures in vitro in the presence of Mg2+. Thus, genomic MMTV promoter chromatin retains the ability to form classical higher-order structures under physiological salt conditions, even after dissociation of H1 and binding of several transcription factors and multiprotein complexes. These results suggest that transcriptionally active eukaryotic promoters may function in a locally folded chromatin environment in vivo.

Acetylation↗

PubMed2003

NSD1 is essential for early post-implantation development and has a catalytically active SET domain.

The nuclear receptor-binding SET domain-containing protein (NSD1) belongs to an emerging family of proteins, which have all been implicated in human malignancy. To gain insight into the biological functions of NSD1, we have generated NSD1-deficient mice by gene disruption. Homozygous mutant NSD1 embryos, which initiate mesoderm formation, display a high incidence of apoptosis and fail to complete gastrulation, indicating that NSD1 is a developmental regulatory protein that exerts function(s) essential for early post-implantation development. We have also examined the enzymatic potential of NSD1 and found that its SET domain possesses intrinsic histone methyltransferase activity with specificity for Lys36 of histone H3 (H3-K36) and Lys20 of histone H4 (H4-K20).

Amino Acid Sequence↗

PubMed2002

Dynamic shuttling and intranuclear mobility of nuclear hormone receptors.

We expressed green fluorescent protein (GFP) chimeras of estrogen, retinoic acid, and thyroid hormone receptors (ERs, RARs, and TRs, respectively) in HeLa cells to examine nucleocytoplasmic shuttling and intranuclear mobility of nuclear hormone receptors (NRs) by confocal microscopy. These receptors were predominantly in the nucleus and, interestingly, underwent intranuclear reorganization after ligand treatment. Nucleocytoplasmic shuttling was demonstrated by heterokaryon experiments and energy-dependent blockade of nuclear import and leptomycin-dependent blockade of nuclear export. Ligand addition decreased shuttling by GFP-ER, whereas heterodimerization with retinoid X receptor helped maintain TR and RAR within the nucleus. Intranuclear mobility of the GFP-NRs was studied by fluorescence recovery after photo-bleaching +/- cognate ligands. Both GFP-TR and GFP-RAR moved rapidly in the nucleus, and ligand binding did not significantly affect their mobility. In contrast, estrogen binding decreased the mobility of GFP-ER and also increased the fraction of GFP-ER that was unable to diffuse. These effects were even more pronounced with tamoxifen. Co-transfection of the co-activator, SRC-1, further slowed the mobility of liganded GFP-ER. Our findings suggest estradiol and tamoxifen exert differential effects on the intranuclear mobility of GFP-ER. They also show that ligand-binding and protein-protein interactions can affect the intracellular mobility of some NRs and thereby may contribute to their biological activity.

Cell Nucleus↗

PubMed2002

Dynamic behavior of transcription factors on a natural promoter in living cells.

Through the use of photobleaching techniques, we examined the dynamic interaction of three members of the transcription apparatus with a target promoter in living cells. The glucocorticoid receptor (GR) interacting protein 1 (GRIP-1) exhibits a half maximal time for fluorescent recovery (tau(R)) of 5 s, reflecting the same rapid exchange as observed for GR. In contrast, the large subunit (RPB1) of RNA polymerase II (pol II) required 13 min for complete fluorescence recovery, consistent with its function as a processive enzyme. We also observe a complex induction profile for the kinetics of GR-stimulated transcription. Our results indicate that GR and GRIP-1 as components of the activating complex are in a dynamic equilibrium with the promoter, and must return to the template many times during the course of transcriptional activation.

Animals↗

PubMed2002

Molecular cloning and characterization of STAMP1, a highly prostate-specific six transmembrane protein that is overexpressed in prostate cancer.

We have identified a novel gene, six transmembrane protein of prostate 1 (STAMP1), which is largely specific to prostate for expression and is predicted to code for a 490-amino acid six transmembrane protein. Using a form of STAMP1 labeled with green fluorescent protein in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP1 is localized to the Golgi complex, predominantly to the trans-Golgi network, and to the plasma membrane. STAMP1 also localizes to vesicular tubular structures in the cytosol and colocalizes with the early endosome antigen 1 (EEA1), suggesting that it may be involved in the secretory/endocytic pathways. STAMP1 is highly expressed in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Furthermore, STAMP1 expression is significantly lower in the androgen-dependent human prostate xenograft CWR22 compared with the relapsed derivative CWR22R, suggesting that its expression may be deregulated during prostate cancer progression. Consistent with this notion, in situ analysis of human prostate cancer specimens indicated that STAMP1 is expressed exclusively in the epithelial cells of the prostate and its expression is significantly increased in prostate tumors compared with normal glands. Taken together, these data suggest that STAMP1 may have an important role in the normal prostate cell as well as in prostate cancer progression.

Amino Acid Sequence↗

PubMed2002

Glucocorticoid receptor domain requirements for chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter in different nucleoprotein contexts.

The glucocorticoid receptor (GR) contains several activation domains, tau1 (AF-1), tau2, and AF-2, which were initially defined using transiently transfected reporter constructs. Using domain mutations in the context of full-length GR, this study defines those domains required for activation of the mouse mammary tumor virus (MMTV) promoter in two distinct nucleoprotein configurations. A transiently transfected MMTV template with a disorganized, accessible chromatin structure was largely dependent on the AF-2 domain for activation. In contrast, activation of an MMTV template in organized, replicated chromatin requires both domains but has a relatively larger dependence on the tau1 domain. Domain requirements for GR-induced chromatin remodeling of the latter template were also investigated. Mutation of the AF-2 helix 12 domain partially inhibits the induction of nuclease hypersensitivity, but the inhibition was relieved in the absence of tau1, suggesting the occurrence of an important interaction between the two domains. Further mutational analysis indicates that GR-induced chromatin remodeling requires the ligand-binding domain in the region of helix 3. Our study shows that the GR activation surfaces required for transcriptional modulation of a target promoter were determined in part by its chromatin structure. Within a particular cellular environment the GR appears to possess a significant degree of versatility in the mechanism by which it activates a target promoter.

Alanine↗

PubMed2002

Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin.

We have found that mica surfaces functionalized with aminopropyltriethoxysilane and aldehydes bind chromatin strongly enough to permit stable and reliable solution imaging by atomic force microscopy. The method is highly reproducible, uses very small amounts of material, and is successful even with very light degrees of surface modification. This surface is far superior to the widely used aminopropyltriethoxysilane-derivatized mica surface and permits resolution of structure on the nanometer-scale in an aqueous environment, conditions that are particularly important for chromatin studies. For example, bound nucleosomal arrays demonstrate major structural changes in response to changes in solution conditions, despite their prior fixation (to maintain nucleosome loading) and tethering to the surface with glutaraldehyde. By following individual molecules through a salt titration in a flow-through cell, one can observe significant changes in apparent nucleosome size at lower [salt] and complete loss of DNA from the polynucleosomal array at high salt. The latter result demonstrates that the DNA component in these arrays is not constrained by the tethering. The former result is consistent with the salt-induced loss of histones observed in bulk solution studies of chromatin and demonstrates that even histone components of the nucleosome are somewhat labile in these fixed and tethered arrays. We foresee many important applications for this surface in future atomic force microscopy studies of chromatin.

Aluminum Silicates↗

PubMed2002

Protein dynamics in the nuclear compartment.

The classic view of a transcriptional initiation complex is that of an assembly of factors with many protein-protein contacts, leading to a multi-component complex whose existence is a result of the stabilizing influence of the many intermolecular interactions. Recent findings from protein mobility experiments in living cells indicate that many kinds of nuclear factors move rapidly and exchange quickly with multiple targets. Two countervailing views of factor/regulatory site interactions emerge from the current literature.

Animals↗

PubMed2002

Recruitment of dioxin receptor to active transcription sites.

The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that heterodimerizes with the AhR nuclear translocator (ARNT/HIF-1beta) to form an AhR/ARNT transcription factor complex. This complex binds to specific DNA sites in the regulatory domains of numerous target genes and mediates the biological effects of exogenous ligands. Herein, we have investigated the subcellular distribution of the AhR/ARNT complex in response to ligand stimulation, by using live-cell confocal and high-resolution deconvolution microscopy. We found that unliganded AhR shows a predominantly cytoplasmic diffuse distribution in mouse hepatoma cells. On addition of ligand, AhR rapidly translocates to the nucleus and accumulates in multiple bright foci. Inhibition of transcription prevented the formation of AhR foci. Dual- and triple-immunolabeling experiments, combined with labeling of nascent RNA, showed that the foci are transcription sites, indicating that upon ligand stimulation, AhR is recruited to active transcription sites. The interaction of AhR with ARNT was both necessary and sufficient for the recruitment of AhR to transcription sites. These results indicate that AhR/ARNT complexes are recruited to specific subnuclear compartments in a ligand-dependent manner and that these foci represent the sites of AhR target genes.

Amanitins↗

PubMed2002

ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling.

Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

Adenosine Triphosphate↗

PubMed2002

Selective intranuclear redistribution of PPAR isoforms by RXR alpha.

The intracellular localization of transcriptionally active green fluorescent protein (GFP) chimeras linked to PPARs for human PPAR alpha (GFP-PPARh alpha) and mouse PPAR alpha, beta, and gamma 1 (GFP-PPARm alpha, GFP-PPARm beta, and GFP-PPARm gamma, respectively) was examined in the mouse hepatoma cell line, Hepa-1, using fluorescence microscopy. A predominantly nuclear and diffuse distribution of each isoform was found in both the presence and absence of specific ligands for each receptor. GFP-PPARm alpha-G (containing a Glu282Gly substitution of PPARm alpha) and a phosphorylation mutant, GFP-PPARm gamma-A (containing a Ser82Ala substitution of PPARm gamma), exhibited altered transcriptional activities, but displayed similar intracellular localization patterns compared with their respective wild-type receptors. Coexpression of nuclear receptor corepressor suppressed, whereas steroid receptor coactivator-1 enhanced the transcriptional activity of each of the GFP-PPAR isoforms, but did not discernibly alter their intracellular distributions, both in the presence and absence of PPAR ligands. Interestingly, coexpression of the obligate heterodimeric partner of PPARs, RXR alpha, resulted in an intranuclear redistribution of the GFP-PPARm gamma isoform characterized by a reticulated pattern of the green fluorescent label for PPAR gamma within the nucleus, but not in nucleoli, and a heightened concentration of the fluorescent label surrounding nucleolar structures and at the nuclear membrane. Conversely, coexpression of yellow fluorescent protein-RXR alpha and native PPARm gamma resulted in a similar distribution of the yellow fluorescent tag. This localization pattern was not discernibly altered by PPAR gamma or RXR alpha-specific ligands. These results implicate RXR alpha in the nuclear reorganization of PPAR gamma and suggest that PPAR gamma colocalizes with RXR alpha at specific locations within the nucleus independent of added ligand.

Active Transport, Cell Nucleus↗