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EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

Owen LA, Kowalewski AA, Lessnick SL - PLoS ONE (2008)

Bottom Line: One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature.Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable.Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America.

ABSTRACT
EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

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Transcriptional repression and DNA binding domains are required for NKX2.2-mediated Ewing's sarcoma cell oncogenic transformation.(A) Schematic of wild type and 3x-FLAG tagged NKX2.2 constructs. The positions of the transcriptional repressor domain (TN), the homeodomain (HD), the NK2-specific domain (SD), and the transcriptional activation domain (TAD) are shown. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with the indicated RNAi and cDNA constructs, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control). The positions of endogenous NKX2.2 (end. NKX2.2) and of the exogenous NKX2.2 constructs (exog. NKX2.2) are indicated.
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pone-0001965-g001: Transcriptional repression and DNA binding domains are required for NKX2.2-mediated Ewing's sarcoma cell oncogenic transformation.(A) Schematic of wild type and 3x-FLAG tagged NKX2.2 constructs. The positions of the transcriptional repressor domain (TN), the homeodomain (HD), the NK2-specific domain (SD), and the transcriptional activation domain (TAD) are shown. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with the indicated RNAi and cDNA constructs, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control). The positions of endogenous NKX2.2 (end. NKX2.2) and of the exogenous NKX2.2 constructs (exog. NKX2.2) are indicated.

Mentions: We recently demonstrated that expression of the transcription factor NKX2.2 is upregulated by EWS/FLI in Ewing's sarcoma and is required for the oncogenic phenotype of the disease [8], [15], [16], [17]. In addition to its DNA binding homeodomain (HD), NKX2.2 harbors both transcriptional activation and repression domains, the presence of which suggests that NKX2.2 acts as a transcriptional activator in some contexts, and as a transcriptional repressor in others (Figure 1A; refs. 18,19). Because a role for NKX2.2 in oncogenesis has only recently been reported, we now report on its molecular mechanism in Ewing's sarcoma development.


EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

Owen LA, Kowalewski AA, Lessnick SL - PLoS ONE (2008)

Transcriptional repression and DNA binding domains are required for NKX2.2-mediated Ewing's sarcoma cell oncogenic transformation.(A) Schematic of wild type and 3x-FLAG tagged NKX2.2 constructs. The positions of the transcriptional repressor domain (TN), the homeodomain (HD), the NK2-specific domain (SD), and the transcriptional activation domain (TAD) are shown. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with the indicated RNAi and cDNA constructs, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control). The positions of endogenous NKX2.2 (end. NKX2.2) and of the exogenous NKX2.2 constructs (exog. NKX2.2) are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291578&req=5

pone-0001965-g001: Transcriptional repression and DNA binding domains are required for NKX2.2-mediated Ewing's sarcoma cell oncogenic transformation.(A) Schematic of wild type and 3x-FLAG tagged NKX2.2 constructs. The positions of the transcriptional repressor domain (TN), the homeodomain (HD), the NK2-specific domain (SD), and the transcriptional activation domain (TAD) are shown. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with the indicated RNAi and cDNA constructs, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control). The positions of endogenous NKX2.2 (end. NKX2.2) and of the exogenous NKX2.2 constructs (exog. NKX2.2) are indicated.
Mentions: We recently demonstrated that expression of the transcription factor NKX2.2 is upregulated by EWS/FLI in Ewing's sarcoma and is required for the oncogenic phenotype of the disease [8], [15], [16], [17]. In addition to its DNA binding homeodomain (HD), NKX2.2 harbors both transcriptional activation and repression domains, the presence of which suggests that NKX2.2 acts as a transcriptional activator in some contexts, and as a transcriptional repressor in others (Figure 1A; refs. 18,19). Because a role for NKX2.2 in oncogenesis has only recently been reported, we now report on its molecular mechanism in Ewing's sarcoma development.

Bottom Line: One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature.Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable.Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America.

ABSTRACT
EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

Show MeSH
Related in: MedlinePlus