scholarly journals Elf-1 Contributes to the Function of the Complex Interleukin (IL)-2–responsive Enhancer in the Mouse IL-2 Receptor α Gene

1997 ◽  
Vol 185 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
Irina Serdobova ◽  
Maria Pla ◽  
Patrick Reichenbach ◽  
Peter Sperisen ◽  
Jacques Ghysdael ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Proximal Region ◽  
Binding Motif ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Ets Family ◽  
Cd8 Cell ◽  
Promoter Proximal Region

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2Rα gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2Rα gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2–responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4−CD8− cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4−CD8− thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti–Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

scholarly journals Structure of a Polycomb Response Element and In Vitro Binding of Polycomb Group Complexes Containing GAGA Factor

2000 ◽  
Vol 20 (9) ◽  
pp. 3187-3197 ◽  
Author(s):  
Béatrice Horard ◽  
Christophe Tatout ◽  
Sylvain Poux ◽  
Vincenzo Pirrotta
Keyword(s):  
Polycomb Group ◽  
Gaga Factor ◽  
Consensus Sequences ◽  
Polycomb Response Element ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Polycomb Response Elements ◽  
Polycomb Group Complexes

ABSTRACT Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

scholarly journals HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

2007 ◽  
Vol 27 (12) ◽  
pp. 4207-4216 ◽  
Author(s):  
Smarajit Bandyopadhyay ◽  
Mohammad Z. Ashraf ◽  
Pamela Daher ◽  
Philip H. Howe ◽  
Paul E. DiCorleto
Keyword(s):  
Transcriptional Activation ◽  
Leukocyte Adhesion ◽  
Regulatory Element ◽  
Proximal Region ◽  
Binding Motif ◽  
Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Tnf Α

ABSTRACT The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-α)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp −210 to −221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-α-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-α-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.

Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins

Development ◽  
2000 ◽  
Vol 127 (1) ◽  
pp. 155-166 ◽  
Author(s):  
E. Ferretti ◽  
H. Marshall ◽  
H. Popperl ◽  
M. Maconochie ◽  
R. Krumlauf ◽  
...  
Keyword(s):  
Hox Genes ◽  
Homeodomain Protein ◽  
Homeodomain Proteins ◽  
Specific Expression ◽  
Hox Proteins ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Close Proximity

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Hoxal and Hoxbl. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. We report here that another family of homeodomain proteins (TALE, Three-Amino acids-Loop-Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4-restricted expression. We show that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2, but not Hoxb1, enhancer is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prepl-Pbx and formation and binding of a ternary Hoxbl-Pbxla (or 1b)-Prepl complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxbl and also contains Pbx with either Prepl or Meisl. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs.

scholarly journals MOT1 Can Activate Basal Transcription In Vitro by Regulating the Distribution of TATA Binding Protein between Promoter and Nonpromoter Sites

1999 ◽  
Vol 19 (4) ◽  
pp. 2835-2845 ◽  
Author(s):  
Tamara A. Muldrow ◽  
Allyson M. Campbell ◽  
P. Anthony Weil ◽  
David T. Auble
Keyword(s):  
Transcriptional Control ◽  
Binding Protein ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Wild Type ◽  
Basal Transcription ◽  
Nuclear Extracts ◽  
Transcription In Vitro

ABSTRACT MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis. Consistent with this observation, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo. To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1. Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP. Surprisingly, MOT1 can weakly activate basal transcription in vitro. This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP. With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters. In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1. These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.

scholarly journals Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex

2008 ◽  
Vol 191 (3) ◽  
pp. 762-772 ◽  
Author(s):  
Simon E. S. Bailey ◽  
Finbarr Hayes
Keyword(s):  
Transcriptional Control ◽  
Binding Motif ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
Short Repeat ◽  
Arginine Residues ◽  
And Function ◽  
Operator Site

ABSTRACT YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5′ of yefM-yoeB in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The yefM-yoeB operator site arrangement is exemplified in Escherichia coli: a pair of palindromes with core hexamer motifs and a center-to-center distance of 12 bp overlap the yefM-yoeB promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

scholarly journals Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

2021 ◽  
Author(s):  
Naresh Damuka ◽  
Miranda Orr ◽  
Paul W. Czoty ◽  
Jeffrey L. Weiner ◽  
Thomas J. Martin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Ex Vivo ◽  
Neuroblastoma Cells ◽  
Proper Function ◽  
Brain Functions ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

scholarly journals In vivo tumour imaging employing regional delivery of novel gallium radiolabelled polymer composites

2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Ross W. Stephens ◽  
Gregory D. Tredwell ◽  
Jessica L. Bell ◽  
Karen J. Knox ◽  
Lee A. Philip ◽  
...  
Keyword(s):  
Normal Liver ◽  
Ct Imaging ◽  
Liver Imaging ◽  
Polymer Microspheres ◽  
Tumour Imaging ◽  
Planar Imaging ◽  
Rabbit Lung ◽  
Vitro Binding

Abstract Background Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. Results Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 μm diameter MS than with 30 μm MS, and the smaller microspheres provided more distinct and localised tumour imaging. Conclusion This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

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