scholarly journals PRDM9 forms an active trimer mediated by its repetitive zinc finger array

2018 ◽  
Author(s):  
Theresa Schwarz ◽  
Yasmin Striedner ◽  
Karin Haase ◽  
Jasmin Kemptner ◽  
Nicole Zeppezauer ◽  
...  
Keyword(s):  
Strand Break ◽  
Specific Protein ◽  
Binding Studies ◽  
Recombination Hotspots ◽  
C Terminus ◽  
Dna Binding Site ◽  
Vitro Binding ◽  
Meiotic Initiation

PRDM9 has been identified as a meiosis-specific protein that plays a key role in determining the location of meiotic recombination hotspots. Although it is well-established that PRDM9 is a trans-acting factor directing the double strand break machinery necessary for recombination to its DNA binding site, the details of PRDM9 binding and complex formation are not well known. It has been suggested in several instances that PRDM9 acts as a multimer in vivo; however, there is little understanding about the protein stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and mass spectrometry to characterize the size of the PRDM9 multimer within the active DNA-protein complex of two different murine PRDM9 alleles, PRDM9Cst and PRDM9Dom2. For this purpose, we developed a strategy to infer the molecular weight of the PRDM9-DNA complex from native gel electrophoresis based on gel shift assays (EMSAs). Our results show that PRDM9 binds as a trimer with the DNA. This multimerization is catalysed by the long ZnF array (ZnF) at the C-terminus of the protein and 11, 10, 7 or 5 ZnFs are already sufficient to form a functional trimer. Finally, we also show that only one ZnF-array within the PRDM9 trimer actively binds to the DNA, while the remaining two ZnF-arrays likely maintain the multimer by ZnF-ZnF interactions. Our results have important implications in terms of PRDM9 dosage, which determines the number of active hotspots in meiotic cells, and contribute to elucidate the molecular interactions of PRDM9 with other components of the meiotic initiation machinery.

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation

1987 ◽  
Vol 7 (9) ◽  
pp. 3252-3259
Author(s):  
T Prezant ◽  
K Pfeifer ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Binding Studies ◽  
Multiple Factors ◽  
Vitro Binding ◽  
Genes Encoding ◽  
Nonfermentable Carbon Source

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

scholarly journals Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537 ◽  
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

scholarly journals Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4415-4425 ◽  
Author(s):  
H Chin ◽  
N Nakamura ◽  
R Kamiyama ◽  
N Miyasaka ◽  
JN Ihle ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Interleukin 3 ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Docking Sites ◽  
Carboxy Terminal

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL-3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.

scholarly journals Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

2004 ◽  
Vol 78 (12) ◽  
pp. 6459-6468 ◽  
Author(s):  
Jeffrey S. Johnson ◽  
Yvonne N. Osheim ◽  
Yuming Xue ◽  
Margaux R. Emanuel ◽  
Peter W. Lewis ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Function Analysis ◽  
Specific Protein ◽  
In Vitro Translation ◽  
Major Protein ◽  
Lampbrush Chromosomes ◽  
Binding Studies

ABSTRACT Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.

scholarly journals Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.

1993 ◽  
Vol 13 (1) ◽  
pp. 44-56 ◽  
Author(s):  
G A Hartzog ◽  
R M Myers
Keyword(s):  
Binding Site ◽  
Site Selection ◽  
Selection Procedure ◽  
Beta Globin ◽  
Binding Studies ◽  
Cis Acting ◽  
Vitro Binding ◽  
Relative Potential

Adult beta-globin-like promoters contain a cis-acting element, CCACACCC, that is conserved across species and is required for wild-type levels of transcription. We have studied the contribution of this element and proteins that interact with it to activate beta-globin transcription. We found that an erythroid-like cell line, MEL, contains several proteins that specifically bind the CACCC element. By comparing the DNA-binding properties of promoters with mutations in the CACCC element with the transcriptional activities of these mutant promoters, we found that two CACCC-binding proteins did not bind to mutant promoters that direct decreased levels of transcription. One of these proteins is the transcriptional activator Sp1, and the other we have designated CACD (CACCC-binding species D). We subjected CACD to a binding site selection procedure and obtained high-affinity CACD binding sites that are identical to that of the beta-globin CACCC element. This result, combined with our finding that CACD binds the CACCC element with a higher affinity than does Sp1, argues that the CACCC element is a target of CACD rather than Sp1. The strategy of correlating the results of a binding site selection experiment with those of in vivo expression and in vitro binding studies may allow evaluation of the relative potential of different proteins to activate transcription through a single cis-acting site.

Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

scholarly journals A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 2795-2804 ◽  
Author(s):  
Marcel van Lith ◽  
Anna-Riikka Karala ◽  
Dave Bown ◽  
John A. Gatehouse ◽  
Lloyd W. Ruddock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Germ Cells ◽  
Specific Protein ◽  
Developmentally Regulated ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Disulfide Isomerases ◽  
Chaperone Complex

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

Development of anti-CD30 radioimmunoconstructs (RICs) for treatment of Hodgkin's lymphoma

2010 ◽  
Vol 49 (03) ◽  
pp. 97-105 ◽  
Author(s):  
S. M. Börner ◽  
T. Fischer ◽  
H. Hansen ◽  
R. Schnell ◽  
B. Zimmermanns ◽  
...  
Keyword(s):  
Binding Studies ◽  
High Background ◽  
Plot Analysis ◽  
Biodistribution Studies ◽  
Vitro Binding ◽  
Chloramine T ◽  
Radiochemical Yields ◽  
Tumour Blood

Summary Objectives: Comparison of the binding affinity to a CD30-positive Hodgkin lymphoma (HL) cell line and biodistribution in HL bearing mice of new anti-CD30 radioimmunoconjugates (RICs) of varying structure and labelling nuclides. Methods: The antibodies Ki-4 and 5F11 were radioiodinated by the chloramine T method or labelled with 111In via p-NCSBenzyl- DOTA. In addition, the Ki-4-dimer was investigated in the iodinated form. The RICs were analyzed for retained immunoreactivity by immunochromatography. In-vitro binding studies were performed on CD30-positive L540 cell lines. For in-vivo biodistribution studies, SCID mice bearing human HL xenografts were injected with the various radioimmunoconjugates. After 24 h, activities in the organs and tumour were measured for all 5 RICs. Tumour-free animals were studied in the same way with 131I- Ki-4 24 h p. i. The three RICs with the highest tumour/background ratios 24 h p.i. (131I-Ki-4, 131I–5F11, 111In-bz- DOTA-Ki-4) were analysed further at 48 h and 72 h. Results: All the RICs were successfully labelled with high specific activities (28–47 TBq/ mmol) and sufficient radiochemical yields (> 80%). Scatchard plot analysis proved high tumour affinity (KD = 20–220 nmol/l). In-vivo tumour accumulation in % of injected dose per g tissue (%ID/g) lay between 2.6 (131I-5F11) and 12.3 % ID/g (131I-Ki-4) with permanently high background in blood. Tumour/blood-ratios of all RICs were below one at all time points. Conclusions: In-vitro tumour cell affinities of all RICs were promising. However, in-vivo biokinetics tested in the mouse model did not meet expectations. This highlights the importance of developing and testing further new anti-CD30 conjugates.

scholarly journals Dynamics of Ankyrin-containing Complexes in Chicken Embryonic Erythroid Cells: Role of Phosphorylation

2001 ◽  
Vol 12 (12) ◽  
pp. 3864-3874 ◽  
Author(s):  
Sourav Ghosh ◽  
John V. Cox
Keyword(s):  
Complex Formation ◽  
Anion Exchanger ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Phosphatase Inhibitors ◽  
In Vitro Binding ◽  
Vitro Binding

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

scholarly journals The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

2006 ◽  
Vol 80 (14) ◽  
pp. 6973-6981 ◽  
Author(s):  
Philomena Ostapchuk ◽  
Mary E. Anderson ◽  
Sharanya Chandrasekhar ◽  
Patrick Hearing
Keyword(s):  
Consensus Sequence ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Specific Protein ◽  
Viral Gene ◽  
Sequence Motif ◽  
Binding Studies ◽  
Viral Dna

ABSTRACT Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis-acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N8-CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral genome did not reduce viral gene expression or viral DNA replication but eliminated the production of infectious virus. We suggest that the L4 22-kDa protein, in conjunction with IVa2, plays a critical role in the recognition of the packaging domain of the Ad genome that leads to viral DNA encapsidation. The L4 22-kDa protein is also involved in recognition of transcription elements of the Ad major late promoter.

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