Robert J. G. Haché

Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor.

ABSTRACT 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 G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. 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 α. 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.

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushings syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non‐transcriptional mechanism mediated through the ligand‐binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPα) expression by potentiating its initial transcriptional activation by C/EBPβ. In the absence of steroid, C/EBPβ associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPα promoter with C/EBPβ and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPα expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand‐binding domain suggests a conserved mechanism relevant to many aspects of steroid‐mediated differentiation.

Glucocorticoid receptor homodimers and glucocorticoid-mineralocorticoid receptor heterodimers form in the cytoplasm through alternative dimerization interfaces.

ABSTRACT 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.

Recruitment of Octamer Transcription Factors to DNA by Glucocorticoid Receptor

ABSTRACT Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, we have studied the consequences of protein-protein binding between GR and Oct-1/2. We have determined that GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiated transcriptional activation by Oct-2 100-fold above the level that could be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlated closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR–Oct-1/2 binding was interrupted by the binding of GR to a GRE. We propose a model for transcriptional cooperativity in which GR–Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.

Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPβ by GCN5

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPβ and C/EBPδ. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPβ. This results in a dramatic enhancement of C/EBPβ-dependent transcription from the C/EBPα promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPβ is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98–102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPβ prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPβ-directed transcription and to potentiate C/EBPβ-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPβ appeared to directly interfere with the interaction of HDAC1 with C/EBPβ, suggesting that PCAF/GCN5-dependent acetylation of C/EBPβ serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase PHOSPHORYLATION OF Ser-527 OF THE RAT GLUCOCORTICOID RECEPTOR

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 ± 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Selective Binding of Steroid Hormone Receptors to Octamer Transcription Factors Determines Transcriptional Synergism at the Mouse Mammary Tumor Virus Promoter

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor α, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.

Phosphorylation of Artemis following irradiation-induced DNA damage

Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin‐sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR‐induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR‐induced phosphorylation is mainly, although not solely, dependent on Ataxia‐telangiectasia‐mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro‐generated Artemis mutants, which present impaired IR‐induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis‐deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA‐PKcs and ATR depending upon the type of DNA damage.

An active nuclear retention signal in the glucocorticoid receptor functions as a strong inducer of transcriptional activation

The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin α/β karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.

A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naïve MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.