The CREBBP Acetyltransferase Is a Haploinsufficient Tumor Suppressor in B-cell Lymphoma.

Inactivating mutations of the CREBBP acetyltransferase are highly frequent in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the two most common germinal center (GC)-derived cancers. However, the role of CREBBP inactivation in lymphomagenesis remains unclear. Here, we show that CREBBP regulates enhancer/super-enhancer networks with central roles in GC/post-GC cell fate decisions, including genes involved in signal transduction by the B-cell receptor and CD40 receptor, transcriptional control of GC and plasma cell development, and antigen presentation. Consistently, Crebbp-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of Crebbp was insufficient to initiate malignant transformation, compound Crebbp-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish CREBBP as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.Significance: Loss-of-function mutations of CREBBP are common and early lesions in FL and DLBCL, suggesting a prominent role in lymphoma initiation. Our studies identify the cellular program by which reduced CREBBP dosage facilitates malignant transformation, and have direct implications for targeted lymphoma therapy based on drugs affecting CREBBP-mediated chromatin acetylation. Cancer Discov; 7(3); 322-37. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Prox1 ablation in hepatic progenitors causes defective hepatocyte specification and increases biliary cell commitment.

The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver's architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.

Genetic interaction between mutations in c-Myb and the KIX domains of CBP and p300 affects multiple blood cell lineages and influences both gene activation and repression.

Adult blood cell production or definitive hematopoiesis requires the transcription factor c-Myb. The closely related KAT3 histone acetyltransferases CBP (CREBBP) and p300 (EP300) bind c-Myb through their KIX domains and mice homozygous for a p300 KIX domain mutation exhibit multiple blood defects. Perplexingly, mice homozygous for the same KIX domain mutation in CBP have normal blood. Here we test the hypothesis that the CBP KIX domain contributes subordinately to hematopoiesis via a genetic interaction with c-Myb. We assessed hematopoiesis in mice bearing compound mutations of c-Myb and/or the KIX domains of CBP and p300, and measured the effect of KIX domain mutations on c-Myb-dependent gene expression. We found that in the context of a p300 KIX mutation, the CBP KIX domain mutation affects platelets, B cells, T cells, and red cells. Gene interaction (epistasis) analysis provides mechanistic evidence that blood defects in KIX mutant mice are consistent with reduced c-Myb and KIX interaction. Lastly, we demonstrated that the CBP and p300 KIX domains contribute to both c-Myb-dependent gene activation and repression. Together these results suggest that the KIX domains of CBP, and especially p300, are principal mediators of c-Myb-dependent gene activation and repression that is required for definitive hematopoiesis.

Inhibition of p300 impairs Foxp3⁺ T regulatory cell function and promotes antitumor immunity.

Forkhead box P3 (Foxp3)(+) T regulatory (T(reg)) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3(+) T(reg) cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3(+) T(reg) cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3(+) T(reg) cells increased T cell receptor-induced apoptosis in T(reg) cells, impaired T(reg) cell suppressive function and peripheral T(reg) cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3(+) T(reg) cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish T(reg) cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

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.

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.

Double null cells reveal that CBP and p300 are dispensable for p53 targets p21 and Mdm2 but variably required for target genes of other signaling pathways.

The histone acetyltransferase coactivators CBP (CREBBP) and p300 (EP300) have more than 400 described protein interaction partners and are implicated in numerous transcriptional pathways. We have shown previously that CBP and p300 double knockout mutations in mouse embryonic fibroblasts (dKO MEFs) result in mixed effects on cAMP-inducible gene expression, with many CREB target genes requiring CBP/p300 for full expression, while others are unaffected or expressed better in their absence. Here we used CBP and p300 dKO MEFs to examine gene expression in response to four other signals: DNA damage (via p53), double-stranded RNA, serum, and retinoic acid. We found that while retinoic acid-inducible gene expression tends to be uniformly dependent on CBP/p300, dsRNA- and serum-inducible genes displayed non-uniform requirements for CBP/p300, with the dsRNA-inducible expression of Ifnb1 (interferon-ß) being particularly dependent on CBP/p300. Surprisingly, the p53-dependent genes Cdkn1a (p21/CIP/WAF) and Mdm2 did not require CBP/p300 for their expression. As with cAMP-responsive CREB targets, we propose that the signal-responsive recruitment of CBP and p300 does not necessarily indicate a requirement for these coactivators at a locus. Rather, target gene context (e.g. DNA sequence) influences the extent to which transcription requires CBP/p300 versus other coactivators, which may not be HATs.

CBP/p300 double null cells reveal effect of coactivator level and diversity on CREB transactivation.

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.

Individual CREB-target genes dictate usage of distinct cAMP-responsive coactivation mechanisms.

CREB is a key mediator of cAMP- and calcium-inducible transcription, where phosphorylation of serine 133 in its Kinase-Inducible Domain (KID) is often equated with transactivation. Phospho-Ser133 is required for CREB to bind the KIX domain of the coactivators CBP and p300 (CBP/p300) in vitro, although the importance of this archetype coactivator interaction for endogenous gene expression is unclear. Here, we show that the CREB interaction with KIX is necessary for only a part of cAMP-inducible transcription and CBP/p300 recruitment. Surprisingly, individual cAMP-inducible genes with CREB bound at their promoters differed in their reliance on KIX and none examined showed complete dependence. Alternatively, we found that arginine 314 (Arg314) in the CREB basic-leucine zipper (bZIP) domain contributed to CBP/p300 recruitment and KIX-independent CREB transactivation function. This implicates Transducer Of Regulated CREB (TORC), an unrelated cAMP-responsive coactivator that binds via Arg314, and which can bind CBP/p300, in these functions. Interestingly, KIX was also required for the full cAMP induction of a gene that did not require CREB. Thus, individual CREB-target gene context dictates the relative contribution of at least two different cAMP-responsive coactivation mechanisms.

Differential role for CBP and p300 CREB-binding domain in motor skill learning.

Cyclic adenosine monophosphate response element binding protein (CREB) binding protein (CBP) and E1A binding protein (p300) are highly homologous transcriptional coactivators with histone acetyltransferase activity. Although CBP and p300 have unique functions in vivo during embryogenesis and hematopoiesis, their functions within the nervous system remain poorly understood. The authors demonstrate that these coactivators have differential roles in motor skill learning. Mice with a mutation in the CREB-binding (KIX) domain of CBP exhibited motor learning deficits. However, mice with the analogous mutation in the KIX domain of p300 showed normal motor learning. Further, CREB knock-out mice exhibited a motor learning deficit similar to that of CBP-KIX mutant mice. These results suggest that the CREB-CBP interaction is more limiting or critical than the CREB-p300 interaction for motor skill learning. Thus, CBP and p300 are genetically distinct at the behavioral level.

Conditional knockout mice reveal distinct functions for the global transcriptional coactivators CBP and p300 in T-cell development.

The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300flox) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300flox and a CBP conditional knockout allele (CBPflox) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4- CD8- double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.

Two transactivation mechanisms cooperate for the bulk of HIF-1-responsive gene expression.

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.

Loss of CBP causes T cell lymphomagenesis in synergy with p27Kip1 insufficiency.

CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.

A transcription-factor-binding surface of coactivator p300 is required for haematopoiesis.

The coactivators CBP (Cre-element binding protein (CREB)-binding protein) and its paralogue p300 are thought to supply adaptor molecule and protein acetyltransferase functions to many transcription factors that regulate gene expression. Normal development requires CBP and p300, and mutations in these genes are found in haematopoietic and epithelial tumours. It is unclear, however, which functions of CBP and p300 are essential in vivo. Here we show that the protein-binding KIX domains of CBP and p300 have nonredundant functions in mice. In mice homozygous for point mutations in the KIX domain of p300 designed to disrupt the binding surface for the transcription factors c-Myb and CREB, multilineage defects occur in haematopoiesis, including anaemia, B-cell deficiency, thymic hypoplasia, megakaryocytosis and thrombocytosis. By contrast, age-matched mice homozygous for identical mutations in the KIX domain of CBP are essentially normal. There is a synergistic genetic interaction between mutations in c-Myb and mutations in the KIX domain of p300, which suggests that the binding of c-Myb to this domain of p300 is crucial for the development and function of megakaryocytes. Thus, conserved domains in two highly related coactivators have contrasting roles in haematopoiesis.