DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.
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...
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 glucocorticoidresponsive 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.The initiation of transcription of genes by RNA polymerase II is usually controlled through complex transcriptional regulatory regions containing multiple binding sites for sequencespecific upstream transcription factors (74, 88). Overall transcriptional responsiveness is determined by cooperative and competitive interactions between the DNA-bound factors that are often dependent upon the precise juxtaposition of DNA binding sites (44). The regulatory potential of individual transcription factors can be influenced indirectly through the manipulation of DNA and/or chromatin structure (6,8,55,88,91). The precise arrangement of the transcription factor binding sites is also important for the cooperative interactions of sequence-specific transcription factors with transcriptional coactivators and the basal transcription machinery (43,53,62,80,94).In many instances, transcriptional cooperativity also correlates with direct protein-protein interactions between individual sequence-specific transcription factors. Often, protein-protein contacts between heterologous factors stabilize DNA binding or alter the sequence specificity of binding. Thus, DNA-bound serum response factor is required for the formation of a ternary complex which includes serum response factor accessory protein 1 (SAP-1) (90); the sequence-specific DNA binding of DNA-dependent protein kinase is mediated through its association with DNA-bound Ku autoantigen (30), and the interaction of herpe...
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