CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the ␣-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ER␣/ SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by cotransfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the ␣-helix A␣1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic ␣-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. CBP1 and p300 interact with a wide range of DNA-binding transcription factors and their cofactors (1, 2). Recruitment of CBP and associated factors permits acetylation and methylation of histones and other proteins at gene promoters, leading to chromatin remodeling, RNA polymerase II recruitment, and transcription. The ability of CBP and p300 to form contacts with multiple diverse factors assembled at gene promoters such as the IFN- enhanceosome, facilitates synergistic activation of transcription (3). Conversely, competition between transcription factors for common binding sites on CBP/p300, which are in limiting concentrations in the nucleus, is likely to be important in negative cross-talk, as observed between nuclear receptors (NRs) and AP-1, NFB, or STAT proteins (4, 5) or hypoxia-inducible factor (HIF1␣) and CITED2 (6). Furthermore, CBP and p300 are important targets in viral infection, as they associate with viral proteins such as adenoviral E1A, SV40 large T antigen, and HTLV Tax (1, 2). Thus, CBP and p300 act as molecular integrators of signal transduction pathways regulating cellular processes such as proliferation, differentiation, apoptosis, and the response to viral infection.The interaction of CBP/p300 with a large number of functionally diverse proteins is facilitated by a series of modular protein-binding domains. These include the cysteine/histidinerich domains CH1 and CH3, also known as TAZ1 and ZZ/TAZ2, which are major sites of protein interaction. The CH3/TA...
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3207 KEY WORDSCBP, CAS/CSE1p, importin, NUP93, HDAC inhibitor, acetyltransferase ACKNOWLEDGEMENTS Report Functional Interaction of CREB Binding Protein (CBP) with Nuclear Transport Proteins and Modulation by HDAC Inhibitors ABSTRACTNuclear transport proteins such as CSE1, NUP93 and Importin-α have recently been shown to be chromatin-associated proteins in yeast, which have unexpected functions in gene regulation. Here we report interactions between the mammalian histone acetyltransferase CBP with nuclear transport proteins CAS (a CSE1 homologue) and Importin-α (Impα) and NUP93. CAS was found to bind the SRC1 interaction domain (SID) of CBP via a leucine-rich motif in the N-terminus of the protein, that is conserved in other SID-binding proteins. Coimmunoprecipitation experiments also revealed that CBP and Impα proteins form a complex. As Impα is a known acetylation target of CBP/p300, and is recycled to the cytoplasm via the exportin CAS, we investigated whether HDAC inhibitors would alter the subcellular localization of these proteins. Treatment of COS-1 cells with the HDAC inhibitors trichostatin A or sodium butyrate resulted in sequestration of Impα in the nuclear envelope, accumulation of CAS in nuclear aggregates, and an increased number of CBP-containing PML bodies per cell. In addition, HDACi treatment appeared to enhance the association of Impα and CBP in coimmunoprecipitation experiments. Our results provide evidence for novel functional interactions between the chromatin modification enzyme CBP and nuclear transport proteins in mammalian cells.
MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RAR2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RAR2 gene. In contrast, MOZ-TIF2 upregulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptormediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZdependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters. Acute myeloid leukemia (AML)3 is associated with recurrent, reciprocal chromosomal translocations in hematopoietic progenitor cells. Genes identified at the breakpoints of these translocations include transcriptional regulators such as the DNA binding transcription factor AML1 and/or genes encoding transcriptional cofactors or chromatin modifiers such as MLL, CBP/p300, MOZ, MORF, and TIF2. Oncogenic fusion proteins resulting from these events are likely to cause both global and promoter-specific alterations in chromatin modification, thus perturbing gene expression programs required for myeloid differentiation, leading to leukemia.Chromosomal translocations that fuse the gene encoding the histone acetyltransferase MOZ (monocytic leukemia zinc finger protein; also known as MYST3 or ZNF220) with genes encoding the nuclear receptor coactivators CBP (CREB-binding protein)/p300 or TIF2 (transcriptional intermediary factor) are associated with M4/M5 subtypes of AML (1-6). Recent studies have confirmed that MOZ-CBP and MOZ-TIF2 fusion proteins have the ability to transform cell lines or recapitulate AML in vivo, and some of the molecular mechanisms involved have been revealed (7-10). Retrovirally expressed MOZ-TIF2 immortalized lineage-deficient murine bone marrow cells in vitro (7,8) and induced acute myeloid leukemia in mice in bone marrow transplant assays (8). The CBP-binding domain (AD1) of MOZ-TIF2 was found to be required to extend the proliferative potential of hematopoietic progenitors in vitro and induce AML in vivo (7,8). Previous results from our laboratory have demonstrated that MOZ-TIF2 interacts directly with CBP in vivo and as a consequence inhibits transcription by p53 and nuclear receptors, in...
The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP–CBPΔ998–1087 had a retarded recovery time in the nucleus, as compared to YFP–CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.
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