Loss of H2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-Related CBP Activator Protein or p400 Complexes

The p400 and SRCAP (Snf2-related CBP activator protein) complexes remodel chromatin by catalyzing deposition of histone H2A.Z into nucleosomes. This remodeling activity has been proposed as a basis for regulation of transcription by these complexes. Transcript levels of p21 or Sp1 mRNAs after knockdown of p400 or SRCAP reveals that each regulates transcription of these promoters differently. In this study, we asked whether deposition of H2A.Z within specific nucleosomes by p400 or SRCAP dictates transcriptional activity. Our data indicates that nucleosome density at specific p21 or Sp1 promoter positions is not altered by the loss of either remodeling complex. However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into all p21 and Sp1 promoter nucleosomes. Thus, H2A.Z deposition is not targeted to specific nucleosomes. These results indicate that the deposition of H2A.Z by the p400 or SRCAP complexes is not sufficient to determine how each regulates transcription. This conclusion is further supported by studies that demonstrate a SRCAPΔATP mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers

Transcriptional tools: Small molecules for modulating CBP KIX-dependent transcriptional activators

Previously it was demonstrated that amphipathic isoxazolidines are able to functionally replace the transcriptional activation domains of endogenous transcriptional activators. In addition, in vitro binding studies suggested that a key binding partner of these molecules is the CREB Binding Protein (CBP), more specifically the KIX domain within this protein. Here we show that CBP plays an essential role in the ability of isoxazolidine transcriptional activation domains to activate transcription in cells. Consistent with this model, isoxazolidines are able to function as competitive inhibitors of the activators MLL and Jun, both of which utilize a binding interaction with KIX to up-regulate transcription. Further, modification of the N2 side chain produced three analogs with enhanced potency against Jun-mediated transcription, although increased cytotoxicity was also observed. Collectively these small KIX-binding molecules will be useful tools for dissecting the role of the KIX domain in a variety of pathological processes. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 17–23, 2011.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78234/1/21548_ftp.pd

The Ets-1 transcription factor controls the development and function of natural regulatory T cells

Regulatory T cells (T reg cells) constitute a population of CD4+ T cells that limits immune responses. The transcription factor Foxp3 is important for determining the development and function of T reg cells; however, the molecular mechanisms that trigger and maintain its expression remain incompletely understood. In this study, we show that mice deficient for the Ets-1 transcription factor (Ets-1−/−) developed T cell–mediated splenomegaly and systemic autoimmunity that can be blocked by functional wild-type T reg cells. Spleens of Ets-1−/− mice contained mostly activated T cells, including Th2-polarized CD4+ cells and had reduced percentages of T reg cells. Splenic and thymic Ets-1−/− T reg cells expressed low levels of Foxp3 and displayed the CD103 marker that characterizes antigen-experienced T reg cells. Thymic development of Ets-1−/− T reg cells appeared intrinsically altered as Foxp3-expressing cells differentiate poorly in mixed fetal liver reconstituted chimera and fetal thymic organ culture. Ets-1−/− T reg cells showed decreased in vitro suppression activity and did not protect Rag2−/− hosts from naive T cell–induced inflammatory bowel disease. Furthermore, in T reg cells, Ets-1 interacted with the Foxp3 intronic enhancer and was required for demethylation of this regulatory sequence. These data demonstrate that Ets-1 is required for the development of natural T reg cells and suggest a role for this transcription factor in the regulation of Foxp3 expression

Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype

Genome-wide assessment of differential roles for p300 and CBP in transcription regulation

Despite high levels of homology, transcription coactivators p300 and CREB binding protein (CBP) are both indispensable during embryogenesis. They are largely known to regulate the same genes. To identify genes preferentially regulated by p300 or CBP, we performed an extensive genome-wide survey using the ChIP-seq on cell-cycle synchronized cells. We found that 57% of the tags were within genes or proximal promoters, with an overall preference for binding to transcription start and end sites. The heterogeneous binding patterns possibly reflect the divergent roles of CBP and p300 in transcriptional regulation. Most of the 16 103 genes were bound by both CBP and p300. However, after stimulation 89 and 1944 genes were preferentially bound by CBP or p300, respectively. Target genes were found to be primarily involved in the regulation of metabolic and developmental processes, and transcription, with CBP showing a stronger preference than p300 for genes active in negative regulation of transcription. Analysis of transcription factor binding sites suggest that CBP and p300 have many partners in common, but AP-1 and Serum Response Factor (SRF) appear to be more prominent in CBP-specific sequences, whereas AP-2 and SP1 are enriched in p300-specific targets. Taken together, our findings further elucidate the distinct roles of coactivators p300 and CBP in transcriptional regulation

Conformational Control of the Binding of the Transactivation Domain of the MLL Protein and c-Myb to the KIX Domain of CREB

The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events

Conserved and Distinct Modes of CREB/ATF Transcription Factor Regulation by PP2A/B56γ and Genotoxic Stress

Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56γ, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors

Emerging roles of the mitogen and stress activated kinases MSK1 and MSK2

Mitogen- and stress-activated kinases (MSK) 1 and 2 are nuclear proteins activated downstream of the ERK1/2 or p38 MAPK pathways. MSKs phosphorylate multiple substrates, including CREB and Histone H3, and their major role is the regulation of specific subsets of Immediate Early genes (IEG). While MSKs are expressed in multiple tissues, their levels are high in immune and neuronal cells and it is in these systems most is known about their function. In immunity, MSKs have predominantly anti-inflammatory roles and help regulate production of the anti-inflammatory cytokine IL-10. In the CNS they are implicated in neuronal proliferation and synaptic plasticity. In this review we will focus on recent advances in understanding the roles of MSKs in the innate immune system and neuronal function