It remains uncertain how the DNA sequence of mammalian genes influences the transcriptional response to extracellular signals. Here, we show that the number of CREB-binding sites (CREs) affects whether the related histone acetyltransferases (HATs) CREB-binding protein (CBP) and p300 are required for endogenous gene transcription. Fibroblasts with both CBP and p300 knocked-out had strongly attenuated histone H4 acetylation at CREB-target genes in response to cyclic-AMP, yet transcription was not uniformly inhibited. Interestingly, dependence on CBP/p300 was often different between reporter plasmids and endogenous genes. Transcription in the absence of CBP/p300 correlated with endogenous genes having more CREs, more bound CREB, and more CRTC2 (a non-HAT coactivator of CREB). Indeed, CRTC2 rescued cAMP-inducible expression for certain genes in CBP/p300 null cells and contributed to the CBP/p300-independent expression of other targets. Thus, endogenous genes with a greater local concentration and diversity of coactivators tend to have more resilient-inducible expression. This model suggests how gene expression patterns could be tuned by altering coactivator availability rather than by changing signal input or transcription factor levels.

The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (DeltaCH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of approximately 35-50% of global HIF-1-responsive gene expression, whereas another HIF transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSA(S)) accounts for approximately 70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription.

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. We previously found that the terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) was inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. We show here that overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells.

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.

In response to decreased cellular oxygen concentrations the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. Here we demonstrate that the mechanism of activation of HIF-1alpha is a multi-step process which includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation was shown to be dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se was not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF-1alpha.

The epigenetic changes of the chromatin represent an attractive molecular substrate for adaptation to the environment. We examined here the role of CREB-binding protein (CBP), a histone acetyltransferase involved in mental retardation, in the genesis and maintenance of long-lasting systemic and behavioural adaptations to environmental enrichment (EE). Morphological and behavioural analyses demonstrated that EE ameliorates deficits associated to CBP deficiency. However, CBP-deficient mice also showed a strong defect in environment-induced neurogenesis and impaired EE-mediated enhancement of spatial navigation and pattern separation ability. These defects correlated with an attenuation of the transcriptional programme induced in response to EE and with deficits in histone acetylation at the promoters of EE-regulated, neurogenesis-related genes. Additional experiments in CBP restricted and inducible knockout mice indicated that environment-induced adult neurogenesis is extrinsically regulated by CBP function in mature granule cells. Overall, our experiments demonstrate that the environment alters gene expression by impinging on activities involved in modifying the epigenome and identify CBP-dependent transcriptional neuroadaptation as an important mediator of EE-induced benefits, a finding with important implications for mental retardation therapeutics.