scholarly journals Fatty Acid Binding Protein FABP5: A Novel Therapeutic Target in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2553-2553
Author(s):  
Metis Hasipek ◽  
Yihong Guan ◽  
Dale Grabowski ◽  
Jaroslaw P. Maciejewski ◽  
Hetty E. Carraway ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Therapeutic Target ◽  
Target Genes ◽  
Binding Protein ◽  
Single Agent ◽  
Model Systems ◽  
Fatty Acid Binding ◽  
Genetic Ablation ◽  
Crabp Ii

Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimer with retinoid X receptors (RXRs) and regulate expression of target genes affecting homeostasis of cellular differentiation and death. The success of RAR activation in the treatment of acute promyelocytic leukemia (APL), particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR in AML cells. RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in a context dependent manner. In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II and cellular levels of RA. In cells, expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ. These two different modes of RA delivery leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to differentiation and growth arrest. However, FABP5 high expressing cells, RA is delivered to PPARβ/δ resulting in survival and proliferation. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML with high FABP5. Here, we report the development of a therapeutic approach using a specific FABP5 inhibitor (iFABP5) that facilitates the delivery of RA to tumor suppressor RAR. Methods: Genetic as well as pharmacologic models in AML cell lines were used to validate the therapeutic target by decoupling PPARβ/δ and RAR/RXR pathways. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects of FABP5 overexpression. The efficacy of small molecule pharmacophore were evaluated in vitro in cell free and cell culture model systems as well as in vivo small animal model systems as a proof of therapeutic concept. Results: We analyzed AML samples from different cohorts (Beat AML and TCGA) for the expression levels of genes involved in RAR and PPARβ/δ pathways. Most of the AML patients have upregulation of pro-survival PPARβ/δ pathway genes including FABP5 and downregulation of differentiating promoting RAR pathway genes including critical RA chaperone CRABP2. On the otherhand, ATRA sensitive APL patients and cell lines have lower expression of FABP5 compared to AML. We established FABP5 as a therapeutic target for ATRA sensitization in AML cell line THP1. Genetic ablation of FABP5 using shRNA sensitizes THP1 cells to ATRA treatment compared to scramble control cells. We used in silico approach to develop a novel small molecule iFABP5 that binds to recombinant FABP5 (Kd= 2x10-7) with a 56-fold higher affinity compared to ATRA (Kd=11x10-6 Mole) in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). Efficacy of iFABP5 as a single agent and in combination with ATRA was evaluated in THP1, HL-60, K562, and OCI-AML5 cells. The effect of the combination was most pronounced in THP-1 cells. The iFABP5 synergizes with ATRA and induces the differentiation at 72 hours as assessed by both CD11b and CD14 levels. Treatment of cells with single agent iFABP5 leads to downregulation of PPARβ/δ target genes and upregulation of RAR target genes consistent with the genetic ablation of shFABP5. THP1 cells stably expressing shRNA targeting FABP5 has significant growth perturbation in a NSG mouse model compared to non-targeting shRNA. Pre-clinical in vivo evaluation of iFABP5 in combination with ATRA is ongoing as a therapeutic proof of concept. Conclusion: The lack of effective ATRA response in AML might be due to an aberrant activation of pro-survival PPARβ/δ pathway, which negatively affects ATRA-regulated gene expression and its antileukemic activity. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy.

scholarly journals Is PPARβ/δa Retinoid Receptor?

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-5 ◽  
Author(s):  
Daniel C. Berry ◽  
Noa Noy
Keyword(s):  
Retinoic Acid ◽  
Fatty Acid Binding Protein ◽  
Nuclear Translocation ◽  
Retinoic Acid Receptor ◽  
Binding Protein ◽  
Retinoid Receptor ◽  
Expression Ratio ◽  
Fatty Acid Binding ◽  
Bona Fide ◽  
Crabp Ii

The broad ligand-binding characteristic of PPARβ/δhas long hampered identification of physiologically-meaningful ligands for the receptor. The observations that the activity of PPARβ/δis supported by fatty acid binding protein 5 (FABP5), which directly delivers ligands from the cytosol to the receptor, suggest thatbona fidePPARβ/δligands both activate the receptor, and trigger the nuclear translocation of FABP5. Using these criteria, it was recently demonstrated that all-trans-retinoic acid (RA), the activator of the classical retinoic acid receptor RAR, also serves as a ligand for PPARβ/δ. Partitioning of RA between its two receptors was found to be regulated by FABP5, which delivers it to PPARβ/δ, and cellular RA binding protein II (CRABP-II), which targets it to RAR. Consequently, RA activates PPARβ/δin cells that display a high FABP5/CRABP-II expression ratio. It remains to be clarified whether compounds other than RA may also serve as endogenous activators for this highly promiscuous protein.

scholarly journals Targeting Antagonists of Retinoic Acid Signaling in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4067-4067
Author(s):  
Metis Hasipek ◽  
Dale Grabowski ◽  
Yihong Guan ◽  
James G Phillips ◽  
Jaroslaw P. Maciejewski ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Board Of Directors ◽  
Cell Lines ◽  
Small Molecule ◽  
Therapeutic Strategy ◽  
Target Genes ◽  
Single Agent ◽  
Advisory Committees ◽  
Qrt Pcr ◽  
Crabp Ii

Abstract Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate homeostasis of cellular growth. The success of RAR modulation in the treatment of acute promyelocytic leukemia (APL) particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR. Recent studies have demonstrated that RA can also activate the peroxisome proliferator-activated receptor β/δ (PPARβ/δ). In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II. In cells expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ (Fig 1). These two different modes of RA delivery due to the different ratio of these two cargos leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to apoptosis, growth arrest, and anticancer activity. However, when FABP5 expression is high, RA is delivered to PPARβ/δ resulting in survival, proliferation, and tumor growth. In both cases, retinoid X receptor (RXR) is the indispensable partner of the nuclear receptor involved. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML. Here we report the development of a therapeutic strategy based on a highly specific FABP5 inhibitor (iFABP5) (Fig 2) that will allow the delivery of RA by CRABP-II to activate tumor suppressor function of RAR and RXR. Methods: An iterative approach of design synthesis and activity were employed to select the most potent hit, iFABP5, for further experiments. Expression levels were analyzed by western blot analysis and qRT-PCR. Colony forming assays were used to analyze iFABP5 activity against AML cell lines. Flow cytometry based cell differentiation assay were performed to assess the efficacy of iFABP5 and ATRA combination treatment. Results: The analysis of TCGA data set revealed that a certain class of AML patients (pts) (Trisomy 8 AML) have low levels of CRABP-II and high levels of FABP5, presumably due to gene duplication, that in part explains the inability of ATRA to induce terminal differentiation in AML cells. To test our hypothesis, we screened AML and APL patient (pt) bone marrow cells and found that a number of AML pts bone marrow have high FABP5 and low CRABP-II protein levels while the ATRA responding APL pts has opposite ratio determined by western blotting. Therefore, low CRABP-II and high FABP5 levels in a subset of AML pts lead to the activation of pro-survival PPARβ/δ pathway that promotes proliferation and opposes the differentiation. We also analyzed AML and APL pt samples along with different AML cell lines for mRNA expression using qRT-PCR. High FABP5 levels were observed in the majority of the AML cell lines. Efficacy of novel small molecule FABP5 inhibitor as a single agent and in combination with ATRA was evaluated in HL-60 cells. The FABP5 inhibitor iFABP5 was found to increase differentiation at 72 hours as assessed by both CD11b and CD14 levels in HL-60 cells. To confirm that iFABP5 is targeting FABP5 and indirectly targeting the PPARβ/δ pathway, levels of RAR and PPARβ/δ target genes were evaluated in the absence and presence of iFABP5. Changes in the gene expression of RAR and PPARβ/δ target genes in the presence and absence of iFABP5 were also examined in shFABP5, shPPARβ/δ, and shCRABP2 versus wild type cells. Conclusion: We demonstrated that a small molecule inhibitor of FABP5 synergizes with ATRA and induces the differentiation in AML cells. High FABP5 levels (mRNA and protein) were observed in the majority of the AML cell lines. Hence, FABP5 can be a therapeutic target in AML. Utilizing virtual screening and structurally guided design, we developed a small molecule FABP5 inhibitor that induces monocytic differentiation as observed by increased CD14 surface expression as a single agent and in combination with ATRA. FABP5 is not only a strong target to treat AML pts but also an excellent approach for developing a novel therapeutic for pts where FABP5 expression and activity is high. Disclosures Maciejewski: Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Carraway:Agios: Consultancy, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau.

scholarly journals Sauna dehydration as a new physiological challenge model for intestinal barrier function

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Fernanda Roca Rubio ◽  
Ulrika Eriksson ◽  
Robert J. Brummer ◽  
Julia König
Keyword(s):  
Fatty Acid ◽  
Intestinal Permeability ◽  
Barrier Function ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Fatty Acid Binding ◽  
Acid Binding Protein

AbstractThe intestinal barrier plays a crucial role in maintaining gut health, and an increased permeability has been linked to several intestinal and extra-intestinal disorders. There is an increasing demand for interventions aimed at strengthening this barrier and for in vivo challenge models to assess their efficiency. This study investigated the effect of sauna-induced dehydration on intestinal barrier function (clinicaltrials.gov: NCT03620825). Twenty healthy subjects underwent three conditions in random order: (1) Sauna dehydration (loss of 3% body weight), (2) non-steroidal anti-inflammatory drug (NSAID) intake, (3) negative control. Intestinal permeability was assessed by a multi-sugar urinary recovery test, while intestinal damage, bacterial translocation and cytokines were assessed by plasma markers. The sauna dehydration protocol resulted in an increase in gastroduodenal and small intestinal permeability. Presumably, this increase occurred without substantial damage to the enterocytes as plasma intestinal fatty acid-binding protein (I-FABP) and liver fatty acid-binding protein (L-FABP) were not affected. In addition, we observed significant increases in levels of lipopolysaccharide-binding protein (LBP), IL-6 and IL-8, while sCD14, IL-10, IFN-ɣ and TNF-α were not affected. These results suggest that sauna dehydration increased intestinal permeability and could be applied as a new physiological in vivo challenge model for intestinal barrier function.

Abstract P347: Retinol-binding Protein 7 (RBP7) is Required for PPARG-mediated Endothelial Protection via Adiponectin

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Chunyan Hu ◽  
Henry L Keen ◽  
Ko-Ting Lu ◽  
Deborah R Davis ◽  
Xuebo Liu ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Rna Sequencing ◽  
Basilar Artery ◽  
Vascular Function ◽  
Carotid Arteries ◽  
Target Genes ◽  
Binding Protein ◽  
Retinol Binding Protein ◽  
Specific Expression ◽  
Fatty Acid Binding

PPARγ protects against endothelial dysfunction by regulation of unknown target genes. One such target, RBP7, an intracellular fatty acid-binding protein, exhibits endothelium-specific expression, but its effect on vascular function remain unknown. We hypothesize that RBP7 is endothelial protective. We examined vascular responses in basilar artery (pressurized myograph) of RBP7-knockout (KO) and wild type (WT) mice fed normal chow (ND) or high fat diet (HFD) for 8 wks. Endothelium-dependent acetylcholine (ACh)-induced relaxation was significantly impaired in HFD-fed KO mice (ACh, 100μM: 33±7% KO vs 83±10% WT, p<0.05), but not in ND-fed groups. This response was ameliorated by pre-incubation with superoxide scavenger tempol (1mM) or PEG-superoxide dismutase (100 U/ml). Mean arterial pressure (measured by radiotelemetry), body weight, hepatic steatosis, fasting glucose, glucose tolerance, and insulin sensitivity were similar in HFD-fed KO and WT mice. To identify targets downstream of RBP7, RNA-Sequencing was performed on carotid arteries from 8-week HFD-fed WT and KO mice as well as ND-fed age-matched littermates. Adiponectin (AdipoQ), a PPARγ target, was increased ~6-fold in HFD-fed WT mice, a response that was markedly blunted in KO mice. RNA sequencing was confirmed by qPCR. There was no difference in plasma AdipoQ. AdipoQ protein is expressed in endothelial cells of carotid arteries and its level of expression was increased in HFD-fed WT but not KO mice (AdipoQ/CD31: 1.14±0.1 WT-HFD vs 0.82±0.1 WT-ND, p<0.05; 0.79±0.1 KO-HFD vs 0.81±0.04 KO-ND). This led us to hypothesize that AdipoQ is involved in RBP7-mediated endothelial protection. Incubation of basilar artery with mouse full-length AdipoQ protein (5 μg/mL, 4 hours) significantly ameliorated endothelial dysfunction (ACh, 100 μM: 56±6% AdipoQ+KO vs 26±3% KO, p<0.05) and blunted carotid artery superoxide production in HFD-fed KO mice. AdipoQ also protects against endothelial dysfunction caused by subpressor Ang-II in KO mice. We conclude that RBP7 protects the endothelium from oxidative stress-induced dysfunction through an AdipoQ-dependent mechanism. Our evidence suggests RBP7 is an essential cofactor for activation of some PPARγ target genes in the endothelium.

scholarly journals Effects of Simvastatin on Retinoic Acid System in Primary Human Endometrial Stromal Cells and in a Chimeric Model of Human Endometriosis

2013 ◽  
Vol 98 (3) ◽  
pp. E463-E471 ◽  
Author(s):  
Anna Sokalska ◽  
MariaPia Anderson ◽  
Jesus Villanueva ◽  
Israel Ortega ◽  
Kaylon L. Bruner-Tran ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Binding Protein ◽  
Inhibitory Effect ◽  
Experimental Models ◽  
Nude Mouse Model ◽  
Endometrial Stromal Cells ◽  
Fatty Acid Binding ◽  
Proliferation And Apoptosis ◽  
Chimeric Model ◽  
Hes Cells

Context: Retinoic acid (RA) may promote survival or apoptosis of cells, depending on the levels of binding proteins: apoptosis-inducing cellular RA binding protein 2 (CRABP2), and cell survival-promoting fatty acid binding protein 5 (FABP5). Increased cellular uptake of retinol and altered actions of RA related to reduced expression of CRABP2 may contribute to the development of endometriosis. Recently statins have been shown to inhibit growth of human endometrial stromal (HES) cells and to reduce the number and size of endometriotic implants in experimental models of this disorder. Objective: The objective of the study was to determine whether effects of simvastatin on HES cells and experimental endometriotic implants are related to the modulation of the RA system. Methods: Effects of simvastatin and RA on proliferation and apoptosis of HES cells were evaluated. Expression of stimulated by RA 6 (STRA6), CRABP2, and FABP5 was determined by real-time PCR and Western blotting. Effects of simvastatin were also evaluated in a nude mouse model of human endometriosis. Results: Simvastatin potentiated an inhibitory effect of RA on growth of HES cells. In HES cells, simvastatin induced expression of STRA6 and CRABP2 but not FABP5. Similarly, simvastatin treatment of nude mice bearing human endometrial xenografts led to an increased expression of CRABP2 and STRA6 proteins in ectopic lesions. Conclusions: Simvastatin interacts with the RA system, inducing the expression of the key protein regulating the uptake of retinol (STRA6) and the expression of apoptosis-promoting CRABP2. These effects may contribute to cooperative apoptosis-inducing effects of simvastatin and RA and support the examination of these compounds in the treatment of endometriosis.

scholarly journals The CACCC-Binding Protein KLF3/BKLF Represses a Subset of KLF1/EKLF Target Genes and Is Required for Proper Erythroid Maturation In Vivo

2012 ◽  
Vol 32 (16) ◽  
pp. 3281-3292 ◽  
Author(s):  
A. P. W. Funnell ◽  
L. J. Norton ◽  
K. S. Mak ◽  
J. Burdach ◽  
C. M. Artuz ◽  
...  
Keyword(s):  
Target Genes ◽  
Binding Protein ◽  
Erythroid Maturation

scholarly journals Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B

1989 ◽  
Vol 262 (3) ◽  
pp. 917-922 ◽  
Author(s):  
R K Singh ◽  
B P Sani ◽  
M I Dawson ◽  
Y F Shealy
Keyword(s):  
Retinoic Acid ◽  
Carboxy Group ◽  
Binding Proteins ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biologically Active ◽  
Amide Bond ◽  
Gel Chromatography ◽  
Fatty Acid Binding ◽  
Sepharose 4B

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.

scholarly journals Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma

2020 ◽  
Vol 117 (48) ◽  
pp. 30670-30678
Author(s):  
Olivera Grbovic-Huezo ◽  
Kenneth L. Pitter ◽  
Nicolas Lecomte ◽  
Joseph Saglimbeni ◽  
Gokce Askan ◽  
...  
Keyword(s):  
Large Scale ◽  
Single Agent ◽  
Model Systems ◽  
Ductal Adenocarcinoma ◽  
In Vivo Model ◽  
Cancer Therapies ◽  
Hsp90 Inhibition ◽  
Dismal Prognosis

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

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