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  • The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP. 22434882

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Acetylation of a conserved lysine residue in the ATP binding pocket of p38 augments its kinase activity during hypertrophy of cardiomyocytes. 21444723

    Like phosphorylation, acetylation of lysine residues within a protein is considered a biologically relevant modification that controls the activity of target proteins. During stress of cells, massive protein acetylation takes place. Here, we show that p38 mitogen-activated protein kinase (MAPK), which controls many biological functions during stress, is reversibly acetylated by PCAF/p300 and HDAC3. We identified two acetylated lysine residues, K152 and K53, located in the substrate binding domain and in the ATP-binding pocket of p38, respectively. Acetylation of lysine 53 enhanced the activity of p38 by increasing its affinity for ATP binding. The enhanced acetylation and activation of p38 were found to be in parallel with reduced intracellular ATP levels in cardiomyocytes under stress, as well as in vivo models of cardiac hypertrophy. Thus, our data show, for the first time, that p38 activity is critically regulated by, in addition to phosphorylation, reversible acetylation of a lysine residue, which is conserved in other kinases, implying the possibility of a similar mechanism regulating their activity.
    Document Type:
    Reference
    Product Catalog Number:
    06-933
    Product Catalog Name:
    Anti-acetyl-Lysine Antibody

  • p300 Acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle. 21389279

    The Forkhead Box O (FOXO) transcription factors regulate diverse cellular processes, and in skeletal muscle are both necessary and sufficient for muscle atrophy. Although the regulation of FOXO by Akt is well evidenced in skeletal muscle, the current study demonstrates that FOXO is also regulated in muscle via the histone acetyltransferase (HAT) activities of p300/CREB-binding protein (CBP). Transfection of rat soleus muscle with a dominant-negative p300, which lacks HAT activity and inhibits endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription from the promoter of a bona fide FOXO target gene, atrogin-1. Conversely, increased HAT activity via transfection of either wild-type (WT) p300 or WT CBP repressed FOXO activation in vivo in response to muscle disuse, and in C2C12 cells in response to dexamethasone and acute starvation. Importantly, manipulation of HAT activity differentially regulated the expression of various FOXO target genes. Cotransfection of FOXO1, FOXO3a, or FOXO4 with the p300 constructs further identified p300 HAT activity to also differentially regulate the activity of the FOXO homologues. Markedly, decreased HAT activity strongly increased FOXO3a transcriptional activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear localization. In summary, this study provides the first evidence to support the acetyltransferase activities of p300/CBP in regulating FOXO signaling in skeletal muscle and suggests that acetylation may be an important mechanism to differentially regulate the FOXO homologues and dictate which FOXO target genes are activated in response to varying atrophic stimuli.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Acetylation of EKLF is essential for epigenetic modification and transcriptional activation of the beta-globin locus. 18710946

    Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the beta-like globin locus chromatin structure and activate transcription of the adult beta-globin gene. By establishing an EKLF-null erythroid line whose closed beta-locus chromatin structure and silent beta-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult beta-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult beta-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Acetylation controls Notch3 stability and function in T-cell leukemia. 22120716

    Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R(1692-1731) mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R(1692-1731) mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.
    Document Type:
    Reference
    Product Catalog Number:
    06-599
    Product Catalog Name:
    Anti-acetyl-Histone H3 Antibody

  • Gli2 acetylation at lysine 757 regulates hedgehog-dependent transcriptional output by preventing its promoter occupancy. 23762415

    The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.
    Document Type:
    Reference
    Product Catalog Number:
    06-933
    Product Catalog Name:
    Anti-acetyl-Lysine Antibody

  • Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity. 22973553

    Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.
    Document Type:
    Reference
    Product Catalog Number:
    06-933
    Product Catalog Name:
    Anti-acetyl-Lysine Antibody

  • Acetylation and deacetylation regulate CCAAT/enhancer binding protein beta at K39 in mediating gene transcription. 18486321

    The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) contains multiple acetylation sites, including lysine (K) 39. Mutation of C/EBPbeta at K39, an acetylation site in the transcriptional activation domain, impairs transcription of C/EBPbeta target genes in a dominant-negative fashion. Further, K39 of C/EBPbeta can be deacetylated by HDAC1, and HDAC1 may decrease C/EBPbeta-mediated transcription, suggesting that acetylation of C/EBPbeta at K39 is dynamically regulated in mediating gene transcription. Acetylation of endogenous C/EBPbeta at K39 is detected in adipose tissue, and also occurs in 3T3-L1 cells undergoing adipocyte conversion. In addition, mutation of K39 in C/EBPbeta impairs activation of its target genes encoding C/EBPalpha and PPARgamma, essential mediators of adipogenesis, as well as adipocyte genes for leptin and Glut4. These findings suggest that acetylation of C/EBPbeta at K39 is an important and dynamic regulatory event that contributes to its ability to transactivate target genes, including those associated with adipogenesis and adipocyte function.
    Document Type:
    Reference
    Product Catalog Number:
    05-515
    Product Catalog Name:
    Anti-acetyl-Lysine Antibody, clone 4G12