This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakagama, H. Articles by Housman, D. E. Search for Related Content PubMed PubMed Citation Articles by Nakagama, H. Articles by Housman, D. E.

 Previous Article  |  Next Article 

Mol. Cell. Biol., 03 1995, 1489-1498, Vol 15, No. 3
Copyright © 1995, American Society for Microbiology

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product

H Nakagama, G Heinrich, J Pelletier and DE Housman
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold- higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.

This article has been cited by other articles:

• Klattig, J., Sierig, R., Kruspe, D., Besenbeck, B., Englert, C. (2007). Wilms' Tumor Protein Wt1 Is an Activator of the Anti-Mullerian Hormone Receptor Gene Amhr2. Mol. Cell. Biol. 27: 4355-4364 [Abstract] [Full Text]  
• Kirschner, K. M., Wagner, N., Wagner, K.-D., Wellmann, S., Scholz, H. (2006). The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the {alpha}4integrin Gene. J. Biol. Chem. 281: 31930-31939 [Abstract] [Full Text]  
• Rong, Y., Cheng, L., Ning, H., Zou, J., Zhang, Y., Xu, F., Liu, L., Chang, Z., Fu, X.-Y. (2006). Wilms' Tumor 1 and Signal Transducers and Activators of Transcription 3 Synergistically Promote Cell Proliferation: A Possible Mechanism in Sporadic Wilms' Tumor. Cancer Res. 66: 8049-8057 [Abstract] [Full Text]  
• Morrison, D. J., English, M. A., Licht, J. D. (2005). WT1 Induces Apoptosis through Transcriptional Regulation of the Proapoptotic Bcl-2 Family Member Bak. Cancer Res. 65: 8174-8182 [Abstract] [Full Text]  
• Discenza, M. T., Pelletier, J. (2004). Insights into the physiological role of WT1 from studies of genetically modified mice. Physiol. Genomics 16: 287-300 [Abstract] [Full Text]  
• Ito, E., Honma, R., Imai, J.-i., Azuma, S., Kanno, T., Mori, S., Yoshie, O., Nishio, J., Iwasaki, H., Yoshida, K., Gohda, J., Inoue, J.-i., Watanabe, S., Semba, K. (2003). A Tetraspanin-Family Protein, T-Cell Acute Lymphoblastic Leukemia-Associated Antigen 1, Is Induced by the Ewing's Sarcoma-Wilms' Tumor 1 Fusion Protein of Desmoplastic Small Round-Cell Tumor. Am. J. Pathol. 163: 2165-2172 [Abstract] [Full Text]  
• Gross, I., Morrison, D. J., Hyink, D. P., Georgas, K., English, M. A., Mericskay, M., Hosono, S., Sassoon, D., Wilson, P. D., Little, M., Licht, J. D. (2003). The Receptor Tyrosine Kinase Regulator Sprouty1 Is a Target of the Tumor Suppressor WT1 and Important for Kidney Development. J. Biol. Chem. 278: 41420-41430 [Abstract] [Full Text]  
• Lam-Yuk-Tseung, S., Govoni, G., Forbes, J., Gros, P. (2003). Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6. Blood 101: 3699-3707 [Abstract] [Full Text]  
• Lee, T. H., Lwu, S., Kim, J., Pelletier, J. (2002). Inhibition of Wilms Tumor 1 Transactivation by Bone Marrow Zinc Finger 2, a Novel Transcriptional Repressor. J. Biol. Chem. 277: 44826-44837 [Abstract] [Full Text]  
• Wilhelm, D., Englert, C. (2002). The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes Dev. 16: 1839-1851 [Abstract] [Full Text]  
• LEE, T. H., PELLETIER, J. (2001). Functional characterization of WT1 binding sites within the human vitamin D receptor gene promoter. Physiol. Genomics 7: 187-200 [Abstract] [Full Text]  
• Aigueperse, C., Val, P., Pacot, C., Darne, C., Lalli, E., Sassone-Corsi, P., Veyssiere, G., Jean, C., Martinez, A. (2001). SF-1 (Steroidogenic Factor-1), C/EBP{beta} (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells. Mol. Endocrinol. 15: 93-111 [Abstract] [Full Text]  
• Hosono, S., Gross, I., English, M. A., Hajra, K. M., Fearon, E. R., Licht, J. D. (2000). E-cadherin Is a WT1 Target Gene. J. Biol. Chem. 275: 10943-10953 [Abstract] [Full Text]  
• Scharnhorst, V., Dekker, P., van der Eb, A. J., Jochemsen, A. G. (2000). Physical Interaction between Wilms Tumor 1 and p73 Proteins Modulates Their Functions. J. Biol. Chem. 275: 10202-10211 [Abstract] [Full Text]  
• Scharnhorst, V., Dekker, P., van der Eb, A. J., Jochemsen, A. G. (1999). Internal Translation Initiation Generates Novel WT1 Protein Isoforms with Distinct Biological Properties. J. Biol. Chem. 274: 23456-23462 [Abstract] [Full Text]  
• McCoy, C., McGee, S. B., Cornwell, M. M. (1999). The Wilms' Tumor Suppressor, WT1, Inhibits 12-O-Tetradecanoylphorbol-13-acetate Activation of the Multidrug Resistance-1 Promoter. Cell Growth Differ. 10: 377-386 [Abstract] [Full Text]  
• English, M. A., Licht, J. D. (1999). Tumor-associated WT1 Missense Mutants Indicate That Transcriptional Activation by WT1 Is Critical for Growth Control. J. Biol. Chem. 274: 13258-13263 [Abstract] [Full Text]  
• Kim, J., Prawitt, D., Bardeesy, N., Torban, E., Vicaner, C., Goodyer, P., Zabel, B., Pelletier, J. (1999). The Wilms' Tumor Suppressor Gene (wt1) Product Regulates Dax-1 Gene Expression during Gonadal Differentiation. Mol. Cell. Biol. 19: 2289-2299 [Abstract] [Full Text]  
• Santiago, F. S., Lowe, H. C., Day, F. L., Chesterman, C. N., Khachigian, L. M. (1999). Early Growth Response Factor-1 Induction by Injury Is Triggered by Release and Paracrine Activation by Fibroblast Growth Factor-2. Am. J. Pathol. 154: 937-944 [Abstract] [Full Text]  
• Liu, C., Yao, J., de Belle, I., Huang, R.-P., Adamson, E., Mercola, D. (1999). The Transcription Factor EGR-1 Suppresses Transformation of Human Fibrosarcoma HT1080 Cells by Coordinated Induction of Transforming Growth Factor-beta 1, Fibronectin, and Plasminogen Activator Inhibitor-1. J. Biol. Chem. 274: 4400-4411 [Abstract] [Full Text]  
• Chun, S.-Y., McGee, E. A., Hsu, S. Y., Minami, S., LaPolt, P. S., Yao, H. H.-C., Bahr, J. M., Gougeon, A., Schomberg, D. W., Hsueh, A. J.W. (1999). Restricted Expression of WT1 Messenger Ribonucleic Acid in Immature Ovarian Follicles: Uniformity in Mammalian and Avian Species and Maintenance during Reproductive Senescence. Biol. Reprod. 60: 365-373 [Abstract] [Full Text]  
• Zhan, Q., Chen, I-T., Antinore, M. J., Fornace, A. J. Jr. (1998). Tumor Suppressor p53 Can Participate in Transcriptional Induction of the GADD45 Promoter in the Absence of Direct DNA Binding. Mol. Cell. Biol. 18: 2768-2778 [Abstract] [Full Text]  
• Smith, S. I., Weil, D., Johnson, G. R., Boyd, A. W., Li, C. L. (1998). Expression of the Wilms' Tumor Suppressor Gene, WT1, Is Upregulated by Leukemia Inhibitory Factor and Induces Monocytic Differentiation in M1 Leukemic Cells. Blood 91: 764-773 [Abstract] [Full Text]  
• Garrett-Sinha, L. A., Eberspaecher, H., Seldin, M. F., de Crombrugghe, B. (1996). A Gene for a Novel Zinc-finger Protein Expressed in Differentiated Epithelial Cells and Transiently in Certain Mesenchymal Cells. J. Biol. Chem. 271: 31384-31390 [Abstract] [Full Text]  
• Biesiada, E., Razandi, M., Levin, E. R. (1996). Egr-1 Activates Basic Fibroblast Growth Factor Transcription. MECHANISTIC IMPLICATIONS FOR ASTROCYTE PROLIFERATION. J. Biol. Chem. 271: 18576-18581 [Abstract] [Full Text]  
• Hewitt, S. M., Fraizer, G. C., Wu, Y.-J., Rauscher, F. J. III, Saunders, G. F. (1996). Differential Function of Wilms' Tumor Gene WT1 Splice Isoforms in Transcriptional Regulation. J. Biol. Chem. 271: 8588-8592 [Abstract] [Full Text]  
• Khachigian, L. M., Williams, A. J., Collins, T. (1995). Interplay of Sp1 and Egr-1 in the Proximal Platelet-derived Growth Factor A-Chain Promoter in Cultured Vascular Endothelial Cells. J. Biol. Chem. 270: 27679-27686 [Abstract] [Full Text]  
• Das, A., Chendil, D., Dey, S., Mohiuddin, M., Mohiuddin, M., Milbrandt, J., Rangnekar, V. M., Ahmed, M. M. (2001). Ionizing Radiation Down-regulates p53 Protein in Primary Egr-1-/- Mouse Embryonic Fibroblast Cells Causing Enhanced Resistance to Apoptosis. J. Biol. Chem. 276: 3279-3286 [Abstract] [Full Text]  
• KIM, J., PELLETIER, J. (1999). Molecular genetics of chromosome translocations involving EWS and related family members. Physiol. Genomics 1: 127-138 [Abstract] [Full Text]