A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

ABSTRACT Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymenamacronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

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Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles

Chemical Effects in Biological Systems (CEBS) ◽

10.22427/ntp-data-002-00060-0001-0000-9 ◽

2018 ◽

Author(s):

Kenneth Korach

Keyword(s):

Gene Regulation ◽

Bisphenol A ◽

Transcriptional Activation ◽

Androgen Receptors

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PCAF Interacts with Tax and Stimulates Tax Transactivation in a Histone Acetyltransferase-Independent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8136 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8136-8145 ◽

Cited By ~ 97

Author(s):

Hua Jiang ◽

Hanxin Lu ◽

R. Louis Schiltz ◽

Cynthia A. Pise-Masison ◽

Vasily V. Ogryzko ◽

...

Keyword(s):

Transcriptional Activation ◽

Histone Acetyltransferase ◽

Point Mutations ◽

Creb Binding Protein ◽

Independent Manner ◽

Cell Leukemia Virus Type ◽

Knock Out ◽

Human T Cell

ABSTRACT Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

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RBP-L, a transcription factor related to RBP-Jkappa.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2679 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2679-2687 ◽

Cited By ~ 83

Author(s):

S Minoguchi ◽

Y Taniguchi ◽

H Kato ◽

T Okazaki ◽

L J Strobl ◽

...

Keyword(s):

Transcriptional Activation ◽

Notch Receptor ◽

In Vitro Assays ◽

Functional Interaction ◽

Protein Protein Interaction ◽

Isolation And Characterization ◽

Signalling Molecule

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

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The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

Journal of Bacteriology ◽

10.1128/jb.185.15.4418-4423.2003 ◽

2003 ◽

Vol 185 (15) ◽

pp. 4418-4423 ◽

Cited By ~ 31

Author(s):

Anne Marie Di Guilmi ◽

Andréa Dessen ◽

Otto Dideberg ◽

Thierry Vernet

Keyword(s):

Streptococcus Pneumoniae ◽

Pathogenic Bacteria ◽

Catalytic Domain ◽

Soluble Form ◽

Lipid Ii ◽

Extracellular Region ◽

Catalytically Active ◽

Bacterial Peptidoglycan

ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

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Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4257-4269.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4257-4269

Author(s):

A Shimamura ◽

D Tremethick ◽

A Worcel

Keyword(s):

Xenopus Laevis ◽

Transcriptional Activation ◽

High Speed ◽

Histone H1 ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

5S Rna ◽

Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

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Human TAFII130 Is a Coactivator for NFATp

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3503-3513.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3503-3513 ◽

Cited By ~ 11

Author(s):

Loree J. Kim ◽

Anita G. Seto ◽

Tuan N. Nguyen ◽

James A. Goodrich

Keyword(s):

Transcriptional Activation ◽

Biochemical Characterization ◽

Luciferase Reporter ◽

Transcription System ◽

Transcription In Vitro ◽

Necessary And Sufficient ◽

In Vitro Interaction ◽

Tfiid Complex

ABSTRACT NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAFII) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAFII130 (hTAFII130) and itsDrosophila melanogaster homolog dTAFII110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAFII130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAFII250, and hTAFII130 supported NFATp-activated transcription, demonstrating the ability of hTAFII130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAFII130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAFII130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.

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Transcriptional Regulation of the MDR1 Gene by Histone Acetyltransferase and Deacetylase Is Mediated by NF-Y

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4377 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4377-4384 ◽

Cited By ~ 214

Author(s):

Shengkan Jin ◽

Kathleen W. Scotto

Keyword(s):

Transcriptional Activation ◽

Histone Acetyltransferase ◽

Binding Protein ◽

Trichostatin A ◽

Present Report ◽

Dominant Negative ◽

Creb Binding Protein ◽

Wild Type ◽

Ccaat Box

ABSTRACT Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level ofMDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

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SPRINT: a Cas13a-based platform for detection of small molecules

Nucleic Acids Research ◽

10.1093/nar/gkaa673 ◽

2020 ◽

Vol 48 (17) ◽

pp. e101-e101 ◽

Cited By ~ 1

Author(s):

Roman S Iwasaki ◽

Robert T Batey

Keyword(s):

Small Molecules ◽

Point Of Care ◽

Rnase Activity ◽

Enzymatic Reactions ◽

Biological Engineering ◽

Detailed Characterization ◽

Specific Effector ◽

Transcriptional Output

Abstract Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.

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Polyomavirus Large T Antigen Binds the Transcriptional Coactivator Protein p300

Journal of Virology ◽

10.1128/jvi.73.2.1734-1739.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1734-1739 ◽

Cited By ~ 15

Author(s):

Maria Nemethova ◽

Erhard Wintersberger

Keyword(s):

Viral Protein ◽

Histone Acetyltransferase ◽

T Antigen ◽

Binding Motif ◽

Large T Antigen ◽

Transcriptional Coactivator ◽

Coactivator Protein ◽

Histone Acetyltransferase Activity

ABSTRACT Using coimmunoprecipitation and glutathioneS-transferase pulldown experiments, we found that polyomavirus large T antigen binds to p300 in vivo and in vitro. The N-terminal region of the viral protein, including the pRB binding motif, was dispensable for this interaction, which involved several regions within the C-terminal half of the large T antigen. Interestingly, anti-T antibody coimmunoprecipitated a subspecies of p300 which has high histone acetyltransferase activity.

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