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Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's 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/EBPa) expression by potentiating its initial transcriptional activation by C/EBPb. In the absence of steroid, C/EBPb 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/EBPa promoter with C/EBPb and promoted the deacetylation of histone H4. Steroid induced the speci®c 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/EBPa 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. Keywords: CAAT enhancer binding protein b function/ histone deacetylase 1/initiation of preadipocyte differentiation/26S proteasome/steroid hormone action IntroductionThe glucocorticoid receptor (GR) is a ligand-activated nuclear hormone receptor that regulates gene expression primarily through direct interaction with DNA response elements (Mangelsdorf et al., 1995). Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing's syndrome (Peeke and Chrousos, 1995). Weight gain is also a sideeffect of immunosuppressive glucocorticoid therapies (Pijl and Meinders, 1996). In rodents, the weight loss that follows adrenalectomy is prevented by glucocorticoid replacement (Freedman et al., 1986;Sainsbury et al., 2001). However, gene-targeted mice in which GR is compromised for DNA binding are of normal weight and do not display any overt signs of alterations in adipogenesis, suggesting that the effects of glucocorticoids on adipogenesis may be mediated through a non-genomic mechanism (Reichardt et al., 2000a).The adipocytes that constitute white fat originate from committed precursor cells, which differentiate in response to a series of cues including insulin and inducers of cAMP. In primary preadipocytes and most cell culture models, glucocorticoids strongly potentiate differentiation (Gregoire et al., 1998). The early responses to insulin and cAMP include the transient induction of CCAAT/enhancer binding protein (C/EBP) b and C/EBPd, and overexpression of C/EBPb is suf®cient to force preadipocyte differentiation in culture (Yeh et al., 1995). In vivo, the stimulatory effect of C/EBPb activity is complemented by the action of C/...

The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to ; radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5V regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress. (Cancer Res 2005; 65(23): 10750-8)

Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein previously implicated in various signaling pathways, but whose function remains enigmatic. Here, we provide evidence that RanBPM functions as an activator of apoptotic pathways induced by DNA damage. First, transient expression of RanBPM in HeLa cells induced cell death through caspase activation, and in the long-term, forced expression of RanBPM impaired cell viability. RanBPM COOH-terminal domain stimulated the ability of RanBPM to induce caspase activation, whereas this activity was negatively regulated by the central SPRY domain. Second, small interfering RNA-directed knockdown of RanBPM prevented DNA damage-induced apoptosis, as evidenced by the marked reduction in caspase-3 and caspase-2 activation. This correlated with a magnitude fold increase in the survival of RanBPM-depleted cells. Following ionizing radiation treatment, we observed a progressive relocalization of RanBPM from the nucleus to the cytoplasm, suggesting that the activation of apoptotic pathways by RanBPM in response to ionizing radiation may be regulated by nucleocytoplasmic trafficking. Finally, RanBPM downregulation was associated with a marked decrease of mitochondria-associated Bax, whereas Bcl-2 overall levels were dramatically upregulated. Overall, our results reveal a novel proapoptotic function for RanBPM in DNA damage-induced apoptosis through the regulation of factors involved in the mitochondrial apoptotic pathway.

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...

The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.

The Ku antigen (70-and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PK cs ) to DNA ends and to specific DNA sequences from which the kinase is activated.

RanBPM (Ran-binding protein M, also called RanBP9) is an evolutionarily conserved, ubiquitous protein which localizes to both nucleus and cytoplasm. RanBPM has been implicated in the regulation of a number of signalling pathways to regulate several cellular processes such as apoptosis, cell adhesion, migration as well as transcription, and plays a critical role during development. In addition, RanBPM has been shown to regulate pathways implicated in cancer and Alzheimer's disease, implying that RanBPM has important functions in both normal and pathological development. While its functions in these processes are still poorly understood, RanBPM has been identified as a component of a large complex, termed the CTLH (C-terminal to LisH) complex. The yeast homologue of this complex functions as an E3 ubiquitin ligase that targets enzymes of the gluconeogenesis pathway. While the CTLH complex E3 ubiquitin ligase activity and substrates still remain to be characterized, the high level of conservation between the complexes in yeast and mammals infers that the CTLH complex could also serve to promote the degradation of specific substrates through ubiquitination, therefore suggesting the possibility that RanBPM's various functions may be mediated through the activity of the CTLH complex.