Paul K. Brindle

The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism

Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.

Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB: CBP-dependent transcriptional activation

Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance both memory and synaptic plasticity. The current model for the action of HDAC inhibitors assumes that they alter gene expression globally and thus affect memory processes in a nonspecific manner. Here, we show that the enhancement of hippocampus-dependent memory and hippocampal synaptic plasticity by HDAC inhibitors is mediated by the transcription factor cAMP response element-binding protein (CREB) and the recruitment of the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CBP) via the CREB-binding domain of CBP. Furthermore, we show that the HDAC inhibitor trichostatin A does not globally alter gene expression but instead increases the expression of specific genes during memory consolidation. Our results suggest that HDAC inhibitors enhance memory processes by the activation of key genes regulated by the CREB:CBP transcriptional complex.

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

B-cell non-Hodgkin’s lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types—follicular lymphoma and diffuse large B-cell lymphoma—harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin’s lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A.

Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. In this study, we test the hypothesis that the stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic subunit (C subunit) into the nucleus. Using fluorescence imaging techniques, we observed that PKA was activated in a stimulus-dependent fashion that led to nuclear entry of C subunit over a 30-min period. The degree of CREB phosphorylation, assessed with antiserum specific for CREB phosphorylated at Ser-133, correlated with the amount of PKA liberated. The time course of phosphorylation closely paralleled the nuclear entry of the catalytic subunit. There was a linear relationship between the subsequent induction of the cAMP-responsive somatostatin gene and the degree of CREB phosphorylation, suggesting that each event--kinase activation, CREB phosphorylation, and transcriptional induction--was tightly coupled to the next. In contrast to other PKA-mediated cellular responses which are rapid and quantitative, the slow, incremental regulation of CREB activity by cAMP suggests that multifunctional kinases like PKA may coordinate cellular responses by dictating the kinetics and stoichiometry of phosphorylation for key substrates like CREB.

Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to cAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.

Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation

Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co‐activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand‐induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF‐mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand‐induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF‐ and CBP/p300‐mediated histone acetylations in gene activation, and suggest an important role of CBP/p300‐mediated H3K18/27ac in NR‐dependent transcription.

CREBBP mutations in relapsed acute lymphoblastic leukaemia

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis–relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

CREB Binding Protein Interacts with Nucleoporin-Specific FG Repeats That Activate Transcription and Mediate NUP98-HOXA9 Oncogenicity

ABSTRACT Genes encoding the Phe-Gly (FG) repeat-containing nucleoporins NUP98 and CAN/NUP214 are at the breakpoints of several chromosomal translocations associated with human acute myeloid leukemia (AML), but their role in oncogenesis is unclear. Here we demonstrate that the NUP98-HOXA9 fusion gene encodes two nuclear oncoproteins with either 19 or 37 NUP98 FG repeats fused to the DNA binding and PBX heterodimerization domains of the transcription factor HOXA9. Both NUP98-HOXA9 chimeras transformed NIH 3T3 fibroblasts, and this transformation required the HOXA9 domains for DNA binding and PBX interaction. Surprisingly, the FG repeats acted as very potent transactivators of gene transcription. This NUP98-derived activity is essential for transformation and can be replaced by the bona fide transactivation domain of VP16. Interestingly, FG repeat-containing segments derived from the nucleoporins NUP153 and CAN/NUP214 functioned similarly to those from NUP98. We further demonstrate that transactivation by FG repeat-rich segments of NUP98 correlates with their ability to interact functionally and physically with the transcriptional coactivators CREB binding protein (CBP) and p300. This finding shows, for the first time, that a translocation-generated fusion protein appears to recruit CBP/p300 as an important step of its oncogenic mechanism. Together, our results suggest that NUP98-HOXA9 chimeras are aberrant transcription factors that deregulate HOX-responsive genes through the transcriptional activation properties of nucleoporin-specific FG repeats that recruit CBP/p300. Indeed, FG repeat-mediated transactivation may be a shared pathogenic function of nucleoporins implicated human AML.

The CREB family of transcription activators.

A diverse family of transcription factors bind to the cAMP-response elements found in a variety of mammalian and viral gene promoters. One of the members of this family, CREB, is being intensively studied so as to elucidate the mechanisms by which second messenger signal transduction pathways act to positively and negatively regulate transcription.

A Role for CREB Binding Protein and p300 Transcriptional Coactivators in Ets-1 Transactivation Functions

ABSTRACT The Ets-1 transcription factor plays a critical role in cell growth and development, but the means by which it activates transcription are still unclear (J. C. Bories, D. M. Willerford, D. Grevin, L. Davidson, A. Camus, P. Martin, D. Stehelin, F. W. Alt, and J. C. Borles, Nature 377:635–638, 1995; N. Muthusamy, K. Barton, and J. M. Leiden, Nature 377:639–642, 1995). Here we show that Ets-1 binds the transcriptional coactivators CREB binding protein (CBP) and the related p300 protein (together referred to as CBP/p300) and that this interaction is required for specific Ets-1 transactivation functions. The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter were repressed by adenovirus E1A, a CBP/p300-specific inhibitor. Furthermore, Ets-1 activity was potentiated by CBP and p300 overexpression. The transactivation function of Ets-1 correlated with its ability to bind an N-terminal cysteine- and histidine-rich region spanning CBP residues 313 to 452. Ets-1 also bound a second cysteine- and histidine-rich region of CBP, between residues 1449 and 1892. Both Ets-1 and CBP/p300 formed a stable immunoprecipitable nuclear complex, independent of DNA binding. This Ets-1–CBP/p300 immunocomplex possessed histone acetyltransferase activity, consistent with previous findings that CBP/p300 is associated with such enzyme activity. Our results indicate that CBP/p300 may mediate antagonistic and synergistic interactions between Ets-1 and other transcription factors that use CBP/p300 as a coactivator, including c-Myb and AP-1.