Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Vrt||WB, IP||Rb||Serum||Polyclonal Antibody|
|Presentation||Rabbit IgG serum containing 0.05% sodium azide and 30% glycerol.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at from date of receipt.|
|Material Size||100 µL|
|Anti-acetyl-Lysine - 2465195||2465195|
|Anti-acetyl-Lysine - 18741||18741|
|Anti-acetyl-Lysine - 19212||19212|
|Anti-acetyl-Lysine - 19702||19702|
|Anti-acetyl-Lysine - 21873||21873|
|Anti-acetyl-Lysine - 26409||26409|
|Anti-acetyl-Lysine - 3188623||3188623|
|Reference overview||Application||Species||Pub Med ID|
|Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases.|
McConnell, MJ; Durand, L; Langley, E; Coste-Sarguet, L; Zelent, A; Chomienne, C; Kouzarides, T; Licht, JD; Guidez, F
Epigenetics & chromatin 8 38 2015
The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood.In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF.Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.
|Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development.|
Karmodiya, K; Anamika, K; Muley, V; Pradhan, SJ; Bhide, Y; Galande, S
Scientific reports 4 6076 2014
In this study, we have investigated genome-wide occurrence of Histone Acetyltransferases (HATs) in genomes of Mus musculus and Danio rerio on the basis of presence of HAT domain. Our study identified a group of proteins that lacks characteristic features of known HAT families, relatively smaller in size and has no other associated domains. Most of the proteins in this unclassified group are Camello proteins, which are not yet known and classified as functional HATs. Our in vitro and in vivo analysis revealed that Camello family proteins are active HATs and exhibit specificity towards histone H4. Interestingly, Camello proteins are among the first identified HATs showing perinuclear localization. Moreover, Camello proteins are evolutionarily conserved in all chordates and are observed for the first time in cnidarians in phylogeny. Furthermore, knockdown of Camello protein (CMLO3) in zebrafish embryos exhibited defects in axis elongation and head formation. Thus, our study identified a novel family of active HATs that is specific for histone H4 acetylation, exhibits perinuclear localization and is essential for zebrafish development.
|Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.|
Heyne, K; Förster, J; Schüle, R; Roemer, K
Nucleic acids research 42 3565-79 2014
NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.
|Myofibrillar Ca(2+) sensitivity is uncoupled from troponin I phosphorylation in hypertrophic obstructive cardiomyopathy due to abnormal troponin T.|
Bayliss, CR; Jacques, AM; Leung, MC; Ward, DG; Redwood, CS; Gallon, CE; Copeland, O; McKenna, WJ; Dos Remedios, C; Marston, SB; Messer, AE
Cardiovascular research 97 500-8 2013
We studied the relationship between myofilament Ca(2+) sensitivity and troponin I (TnI) phosphorylation by protein kinase A at serines 22/23 in human heart troponin isolated from donor hearts and from myectomy samples from patients with hypertrophic obstructive cardiomyopathy (HOCM).We used a quantitative in vitro motility assay. With donor heart troponin, Ca(2+) sensitivity is two- to three-fold higher when TnI is unphosphorylated. In the myectomy samples from patients with HOCM, the mean level of TnI phosphorylation was low: 0.38 ± 0.19 mol Pi/mol TnI compared with 1.60 ± 0.19 mol Pi/mol TnI in donor hearts, but no difference in myofilament Ca(2+) sensitivity was observed. Thus, troponin regulation of thin filament Ca(2+) sensitivity is abnormal in HOCM hearts. HOCM troponin (0.29 mol Pi/mol TnI) was treated with protein kinase A to increase the level of phosphorylation to 1.56 mol Pi/mol TnI. No difference in EC(50) was found in thin filaments containing high and low TnI phosphorylation levels. This indicates that Ca(2+) sensitivity is uncoupled from TnI phosphorylation in HOCM heart troponin. Coupling could be restored by replacing endogenous troponin T (TnT) with the recombinant TnT T3 isoform. No difference in Ca(2+) sensitivity was observed if TnI was exchanged into HOCM heart troponin or if TnT was exchanged into the highly phosphorylated donor heart troponin. Comparison of donor and HOCM heart troponin by mass spectrometry and with adduct-specific antibodies did not show any differences in TnT isoform expression, phosphorylation or any post-translational modifications.An abnormality in TnT is responsible for uncoupling myofibrillar Ca(2+) sensitivity from TnI phosphorylation in the septum of HOCM patients.
|mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases.|
Mitchell, L; Huard, S; Cotrut, M; Pourhanifeh-Lemeri, R; Steunou, AL; Hamza, A; Lambert, JP; Zhou, H; Ning, Z; Basu, A; Côté, J; Figeys, DA; Baetz, K
Proceedings of the National Academy of Sciences of the United States of America 110 E1641-50 2013
Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.
|Gli2 acetylation at lysine 757 regulates hedgehog-dependent transcriptional output by preventing its promoter occupancy.|
Coni, S; Antonucci, L; D'Amico, D; Di Magno, L; Infante, P; De Smaele, E; Giannini, G; Di Marcotullio, L; Screpanti, I; Gulino, A; Canettieri, G
PloS one 8 e65718 2013
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.
|Tasquinimod Is an Allosteric Modulator of HDAC4 survival signaling within the compromised cancer microenvironment.|
Isaacs, JT; Antony, L; Dalrymple, SL; Brennen, WN; Gerber, S; Hammers, H; Wissing, M; Kachhap, S; Luo, J; Xing, L; Björk, P; Olsson, A; Björk, A; Leanderson, T
Cancer research 73 1386-99 2013
Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.
|Modulation of histone deacetylase 6 (HDAC6) nuclear import and tubulin deacetylase activity through acetylation.|
Liu, Y; Peng, L; Seto, E; Huang, S; Qiu, Y
The Journal of biological chemistry 287 29168-74 2012
The reversible acetylation of histones and non-histone proteins by histone acetyltransferases and deacetylases (HDACs) plays a critical role in many cellular processes in eukaryotic cells. HDAC6 is a unique histone deacetylase with two deacetylase domains and a C-terminal zinc finger domain. HDAC6 resides mainly in the cytoplasm and regulates many important biological processes, including cell migration and degradation of misfold proteins. HDAC6 has also been shown to localize in the nucleus to regulate transcription. However, how HDAC6 shuttles between the nucleus and cytoplasm is largely unknown. In addition, it is not clear how HDAC6 enzymatic activity is modulated. Here, we show that HDAC6 can be acetylated by p300 on five clusters of lysine residues. One cluster (site B) of acetylated lysine is in the N-terminal nuclear localization signal region. These lysine residues in site B were converted to glutamine to mimic acetylated lysines. The mutations significantly reduced HDAC6 tubulin deacetylase activity and further impaired cell motility, but had no effect on histone deacetylase activity. More interestingly, these mutations retained HDAC6 in the cytoplasm by blocking the interaction with the nuclear import protein importin-α. The retention of HDAC6 in the cytoplasm by acetylation eventually affects histone deacetylation. Thus, we conclude that acetylation is an important post-translational modification that regulates HDAC6 tubulin deacetylase activity and nuclear import.
|Identifying acetylated proteins in mitosis.|
Carol Chuang,Li-Yuan Yu-Lee
Methods in molecular biology (Clifton, N.J.) 909 2012
Histone deacetylase (HDAC) inhibitors are currently used in anticancer therapy to perturb genomic targets involved in gene transcriptional responses. However, the role of HDAC inhibitors on the acetylation of proteins outside of the transcriptional network has not been thoroughly assessed. We recently discovered that one of the HDACs, HDAC3, is localized on the mitotic spindle and regulates proper mitotic progression (1). To determine potential HDAC targets, we undertook a proteomics approach to search for acetylated proteins in mitosis (2). First, we synchronized cells in mitosis and used a polyclonal anti-acetyl-Lysine antiserum to immunoprecipitate acetylated proteins, followed by their identification by LC-ESI-MS/MS. We then confirmed the acetylation status of several mitotic proteins by anti-acetyl-Lysine immunoprecipitation with a monoclonal antibody followed by Western blot analyses of the proteins of interest. We further confirmed by a reciprocal immunoprecipitation with protein-specific antibody followed by Western blot analysis with another monoclonal anti-acetyl-Lysine antibody. Interestingly, the acetylation of a subset of the mitotic proteins can be further enhanced by treatment with apicidin, a small molecule inhibitor with specificity for HDAC3, suggesting that their acetylation may be regulated by HDAC3 in mitosis. In this chapter, we describe the various techniques using NudC as an example of an acetylated protein that is sensitive to apicidin treatment in mitosis.
|Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.|
Lanzillotta, Annamaria, et al.
Neurobiol. Dis., 49C: 177-189 (2012) 2012
Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. OBJECTIVE: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. METHODS: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). RESULTS: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-x(L) promoter and decreased at the Bim promoter. CONCLUSION: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.