scholarly journals Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and kinetics

2009 ◽  
Vol 419 (1) ◽  
pp. 221-230 ◽  
Author(s):  
Ctirad Hofr ◽  
Pavla Šultesová ◽  
Michal Zimmermann ◽  
Iva Mozgová ◽  
Petra Procházková Schrumpfová ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Telomeric Dna ◽  
Mobility Shift ◽  
Telomere Repeat ◽  
Binding Stoichiometry ◽  
Binding Factor ◽  
Telomere Function ◽  
Telomere Capping ◽  
Mobility Shift Assay ◽  
And Control

Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.

scholarly journals Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

2004 ◽  
Vol 384 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Heiner KOESSLER ◽  
Joerg KAHLE ◽  
Christa BODE ◽  
Detlef DOENECKE ◽  
Werner ALBIG
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Histone H3 ◽  
Maximal Activity ◽  
Significant Loss ◽  
Tata Box ◽  
Mobility Shift ◽  
Nuclear Factor Y ◽  
Binding Factor ◽  
Mobility Shift Assay

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133–227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

scholarly journals Functional interaction of Oct transcription factors with the family of repeats in Epstein–Barr virus oriP

2005 ◽  
Vol 86 (5) ◽  
pp. 1261-1267 ◽  
Author(s):  
J. Almqvist ◽  
J. Zou ◽  
Y. Linderson ◽  
C. Borestrom ◽  
E. Altiok ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Epstein Barr Virus ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
The Family ◽  
Epstein Barr ◽  
Mobility Shift Assay ◽  
Specific Regulation ◽  
Cellular Transcription

The family of repeats (FR) is a major upstream enhancer of the Epstein–Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.

Identification of the human beta-actin enhancer and its binding factor

1988 ◽  
Vol 8 (1) ◽  
pp. 267-272
Author(s):  
T Kawamoto ◽  
K Makino ◽  
H Niwa ◽  
H Sugiyama ◽  
S Kimura ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
High Specificity ◽  
Simian Virus ◽  
Mobility Shift ◽  
Beta Actin ◽  
Binding Factor ◽  
Pair Sequence ◽  
Mobility Shift Assay ◽  
Dyad Symmetry

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

scholarly journals Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein–Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

2009 ◽  
Vol 90 (5) ◽  
pp. 1183-1189 ◽  
Author(s):  
Daniel Salamon ◽  
Ferenc Banati ◽  
Anita Koroknai ◽  
Mate Ravasz ◽  
Kalman Szenthe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cpg Methylation ◽  
Ctcf Binding ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
Mobility Shift Assay

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

NF-κB activation in organs from STZ-treated rats

2011 ◽  
Vol 36 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Marius Locke ◽  
Jocelyn Anderson
Keyword(s):  
Subunit Composition ◽  
Sprague Dawley ◽  
Mobility Shift ◽  
Diabetic State ◽  
Sprague Dawley Rats ◽  
Inflammatory State ◽  
Mobility Shift Assay ◽  
Related Proteins ◽  
And Control ◽  
Insight Into

Nuclear factor kappa B (NF-κB) is a ubiquitously expressed transcription factor comprised of various subunits (p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB, and c-Rel). Activation of certain NF-κB subunits appears to foster an inflammatory state that may promote the development of disease. Thus characterizing the specific NF-κB subunits may provide insight into the pathogenesis of certain diseases. The purpose of this study was to determine if 1 month of a diabetic state, induced by streptozotocin (STZ) treatment, alters the constitutive level of NF-κB activation, its subunit composition, or the content of NF-κB-related proteins in rodent liver, kidney, spleen, and heart. Diabetes was induced in male Sprague–Dawley rats by a single tail vein injection of STZ (55 mg·kg–1 body weight). After 30 days, the heart, liver, spleen, and kidney were assessed for NF-κB activation and subunit composition with electrophoretic mobility shift assay (EMSA), and p50 and p65 subunit content was assessed with Western blotting. In diabetic animals, the constitutive level of NF-κB activation was reduced in liver, but was unchanged in kidney, spleen, and heart. EMSA supershifts showed the predominant subunit in the activated NF-κB complexes from both diabetic and control animals to be p50, although the p65 subunit was detected in NF-κB complexes from diabetic hearts. The content of p50 was unaltered in all diabetic tissues examined, whereas the content of p65 was increased only in hearts from diabetic animals. These findings support the idea that a diabetic state may induce specific changes in NF-κB subunit composition in certain tissues.

scholarly journals Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex.

1996 ◽  
Vol 135 (4) ◽  
pp. 867-881 ◽  
Author(s):  
M E Ludérus ◽  
B van Steensel ◽  
L Chong ◽  
O C Sibon ◽  
F F Cremers ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Nuclear Lamina ◽  
Nuclear Volume ◽  
Fish Analysis ◽  
Telomeric Dna ◽  
Attachment Sites ◽  
Binding Factor ◽  
Telomere Function ◽  
Biochemical Fractionation ◽  
Order Complexes

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

scholarly journals Identification of the human beta-actin enhancer and its binding factor.

1988 ◽  
Vol 8 (1) ◽  
pp. 267-272 ◽  
Author(s):  
T Kawamoto ◽  
K Makino ◽  
H Niwa ◽  
H Sugiyama ◽  
S Kimura ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
High Specificity ◽  
Simian Virus ◽  
Mobility Shift ◽  
Beta Actin ◽  
Binding Factor ◽  
Pair Sequence ◽  
Mobility Shift Assay ◽  
Dyad Symmetry

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

scholarly journals Structural Characterization of Arabidopsis thaliana NAP1-Related Protein 2 (AtNRP2) and Comparison with its Homolog AtNRP1

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2258 ◽  
Author(s):  
Kumar ◽  
Kumar Singh ◽  
Chandrakant Bobde ◽  
Vasudevan
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Titration Calorimetry ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
The Stability ◽  
Structural Architecture ◽  
And Function

Nucleosome Assembly Protein (NAP) is a highly conserved family of histone chaperones present in yeast, animals, and plants. Unlike other organisms, plants possess an additional class of proteins in its NAP family, known as the NAP1-related proteins or NRP. Arabidopsis thaliana possesses two NRP isoforms, namely AtNRP1 and AtNRP2, that share 87% sequence identity. Both AtNRP1 and AtNRP2 get expressed in all the plant tissues. Most works in the past, including structural studies, have focused on AtNRP1. We wanted to do a comparative study of the two proteins to find why the plant would have two very similar proteins and whether there is any difference between the two for their structure and function as histone chaperones. Here we report the crystal structure of AtNRP2 and a comparative analysis of its structural architecture with other NAP family proteins. The crystal structure of AtNRP2 shows it to be a homodimer, with its fold similar to that of other structurally characterized NAP family proteins. Although AtNRP1 and AtNRP2 have a similar fold, upon structural superposition, we find an offset in the dimerization helix of the two proteins. We evaluated the stability, oligomerization status, and histone chaperoning properties of the two proteins, for a comparison. The thermal melting experiments suggest that AtNRP2 is more stable than AtNRP1 at higher temperatures. In addition, electrophoretic mobility shift assay and isothermal titration calorimetry experiments suggest histone binding ability of AtNRP2 is higher than that of AtNRP1. Overall, these results provide insights about the specific function and relevance of AtNRP2 in plants through structural and biophysical studies.

scholarly journals The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein

2007 ◽  
Vol 408 (3) ◽  
pp. 387-393 ◽  
Author(s):  
Yi-Ching Hsieh ◽  
Pei-Jung Tu ◽  
Ying-Yuan Lee ◽  
Chun-Chen Kuo ◽  
Yi-Chien Lin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Mobility Shift ◽  
Telomere Function

Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.

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