Ward Giffin

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.

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.

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.

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

ABSTRACT Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase. While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PKcs) is regulated through its association with Ku is only beginning to be understood. We report that activation of DNA-PKcs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA. Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku–DNA-PKcs. Remarkably, the truncated element was recognized by Ku–DNA-PKcs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PKcs. Analysis of Ku–DNA-PKcs interactions with DNA ends, double- and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PKcs and the activation of kinase activity.

Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3–4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t½ on = 2.0 min) and was exceptionally stable, with an off rate of t½= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins.

DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured single-stranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell.

Differential DNA binding of Ku antigen determines its involvement in DNA replication

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.