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The tumor suppressor gene KCTD11REN is regulated by Sp1 and methylation and its expression is reduced in tumors.

Mancarelli MM, Zazzeroni F, Ciccocioppo L, Capece D, Po A, Murgo S, Di Camillo R, Rinaldi C, Ferretti E, Gulino A, Alesse E - Mol. Cancer (2010)

Bottom Line: A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11).We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events.Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, University of L'Aquila, L'Aquila 67100, Italy.

ABSTRACT
A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells.

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Basal transcriptional activity of human KCTD11 promoter is depending on Sp1 TF. (A) Human KCTD11 promoter was identified by Promoter Inspector http://www.genomatix.de/. Strong matches for Sp1 transcription factor binding sites are boxed. The asterisk indicates the TSS. The CpG dinucleotides are grey highlighted and numbered. (B) Schematic representation of Luc reporter constructs driven by human full-length KCTD11 promoter or deletion mutants. Constructs were generated by PCR using genomic DNA from peripheral blood lymphocytes as template and specific primers [additional file 2] and subcloned in pGL3 basic vector. (C-D) Luciferase assays showing the transcriptional activity of full-length or deletion mutants of KCTD11 promoter. 293T HEK (human embryo kidney) cell line was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma). 2 μg of KCTD11-Luc reporter constructs were transfected using Lipofectamine 2000 (Invitrogen). After 24 hrs, the cells were harvested and firefly luciferase activity was assayed using the Firelite Dual Luminescence Reporter Gene Assay System kit (Perkin-Elmer) normalized to Renilla luciferase activity. Each experiment has been done in triplicates. Values are the means ± S.D.
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Figure 2: Basal transcriptional activity of human KCTD11 promoter is depending on Sp1 TF. (A) Human KCTD11 promoter was identified by Promoter Inspector http://www.genomatix.de/. Strong matches for Sp1 transcription factor binding sites are boxed. The asterisk indicates the TSS. The CpG dinucleotides are grey highlighted and numbered. (B) Schematic representation of Luc reporter constructs driven by human full-length KCTD11 promoter or deletion mutants. Constructs were generated by PCR using genomic DNA from peripheral blood lymphocytes as template and specific primers [additional file 2] and subcloned in pGL3 basic vector. (C-D) Luciferase assays showing the transcriptional activity of full-length or deletion mutants of KCTD11 promoter. 293T HEK (human embryo kidney) cell line was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma). 2 μg of KCTD11-Luc reporter constructs were transfected using Lipofectamine 2000 (Invitrogen). After 24 hrs, the cells were harvested and firefly luciferase activity was assayed using the Firelite Dual Luminescence Reporter Gene Assay System kit (Perkin-Elmer) normalized to Renilla luciferase activity. Each experiment has been done in triplicates. Values are the means ± S.D.

Mentions: To understand the transcriptional regulation of KCTD11, we identified and analyzed the promoter region. Human KCTD11 proximal promoter is a 623 bp region (Fig. 2A). It turned out to be a TATA- and CAAT-less promoter. The transcription start site (TSS) was previously identified [4] (Fig. 2A). Using the TRANSFACT software, we identified six putative binding sites for Sp1 transcription factor (TF), two of them located closely to the TSS (see Fig. 2A, Sp1-E and Sp1-F). Sp1 is a well-characterized transcriptional activator [7,8]. It is essential for proper expression of a large variety of genes involved in development, cell growth regulation and cancer [9,10]. Moreover, Sp1 is responsible for recruiting TATA-binding protein and fixing the TSS at TATAless promoters [9,11]. Thus, the Sp1-E and Sp1-F binding sites on KCTD11 promoter (Fig. 2A) are likely to be responsible for assembling of the basal transcription complex.


The tumor suppressor gene KCTD11REN is regulated by Sp1 and methylation and its expression is reduced in tumors.

Mancarelli MM, Zazzeroni F, Ciccocioppo L, Capece D, Po A, Murgo S, Di Camillo R, Rinaldi C, Ferretti E, Gulino A, Alesse E - Mol. Cancer (2010)

Basal transcriptional activity of human KCTD11 promoter is depending on Sp1 TF. (A) Human KCTD11 promoter was identified by Promoter Inspector http://www.genomatix.de/. Strong matches for Sp1 transcription factor binding sites are boxed. The asterisk indicates the TSS. The CpG dinucleotides are grey highlighted and numbered. (B) Schematic representation of Luc reporter constructs driven by human full-length KCTD11 promoter or deletion mutants. Constructs were generated by PCR using genomic DNA from peripheral blood lymphocytes as template and specific primers [additional file 2] and subcloned in pGL3 basic vector. (C-D) Luciferase assays showing the transcriptional activity of full-length or deletion mutants of KCTD11 promoter. 293T HEK (human embryo kidney) cell line was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma). 2 μg of KCTD11-Luc reporter constructs were transfected using Lipofectamine 2000 (Invitrogen). After 24 hrs, the cells were harvested and firefly luciferase activity was assayed using the Firelite Dual Luminescence Reporter Gene Assay System kit (Perkin-Elmer) normalized to Renilla luciferase activity. Each experiment has been done in triplicates. Values are the means ± S.D.
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Related In: Results  -  Collection

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Figure 2: Basal transcriptional activity of human KCTD11 promoter is depending on Sp1 TF. (A) Human KCTD11 promoter was identified by Promoter Inspector http://www.genomatix.de/. Strong matches for Sp1 transcription factor binding sites are boxed. The asterisk indicates the TSS. The CpG dinucleotides are grey highlighted and numbered. (B) Schematic representation of Luc reporter constructs driven by human full-length KCTD11 promoter or deletion mutants. Constructs were generated by PCR using genomic DNA from peripheral blood lymphocytes as template and specific primers [additional file 2] and subcloned in pGL3 basic vector. (C-D) Luciferase assays showing the transcriptional activity of full-length or deletion mutants of KCTD11 promoter. 293T HEK (human embryo kidney) cell line was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma). 2 μg of KCTD11-Luc reporter constructs were transfected using Lipofectamine 2000 (Invitrogen). After 24 hrs, the cells were harvested and firefly luciferase activity was assayed using the Firelite Dual Luminescence Reporter Gene Assay System kit (Perkin-Elmer) normalized to Renilla luciferase activity. Each experiment has been done in triplicates. Values are the means ± S.D.
Mentions: To understand the transcriptional regulation of KCTD11, we identified and analyzed the promoter region. Human KCTD11 proximal promoter is a 623 bp region (Fig. 2A). It turned out to be a TATA- and CAAT-less promoter. The transcription start site (TSS) was previously identified [4] (Fig. 2A). Using the TRANSFACT software, we identified six putative binding sites for Sp1 transcription factor (TF), two of them located closely to the TSS (see Fig. 2A, Sp1-E and Sp1-F). Sp1 is a well-characterized transcriptional activator [7,8]. It is essential for proper expression of a large variety of genes involved in development, cell growth regulation and cancer [9,10]. Moreover, Sp1 is responsible for recruiting TATA-binding protein and fixing the TSS at TATAless promoters [9,11]. Thus, the Sp1-E and Sp1-F binding sites on KCTD11 promoter (Fig. 2A) are likely to be responsible for assembling of the basal transcription complex.

Bottom Line: A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11).We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events.Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, University of L'Aquila, L'Aquila 67100, Italy.

ABSTRACT
A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells.

Show MeSH
Related in: MedlinePlus