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Functional enhancers at the gene-poor 8q24 cancer-linked locus.

Jia L, Landan G, Pomerantz M, Jaschek R, Herman P, Reich D, Yan C, Khalid O, Kantoff P, Oh W, Manak JR, Berman BP, Henderson BE, Frenkel B, Haiman CA, Freedman M, Tanay A, Coetzee GA - PLoS Genet. (2009)

Bottom Line: Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR).Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness.The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

View Article: PubMed Central - PubMed

Affiliation: USC/Norris Cancer Center, Department of Preventive Medicine, University of Southern California, Los Angeles, CA, USA.

ABSTRACT
Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

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Constitutive enhancer activity of AcH3 peak sequences at 8q24.The DNA sequence containing each of the 15 identified AcH3 sites or a control sequence from the neighboring unacetylated region was inserted upstream of TK-luciferase reporter vector. The constructs were transfected into 5 different cell lines (LNCaP, PC3, HCT 115, COLO 205, and MCF7) along with pRL-TK Renilla luciferase plasmid for 24 h. Dual luciferase assays were conducted. The results were normalized against the internal Renilla control for each transfection. The luciferase activity of the control region was defined as 1. Relative luciferase activity values are presented as mean±SD of triplicate transfections.
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pgen-1000597-g004: Constitutive enhancer activity of AcH3 peak sequences at 8q24.The DNA sequence containing each of the 15 identified AcH3 sites or a control sequence from the neighboring unacetylated region was inserted upstream of TK-luciferase reporter vector. The constructs were transfected into 5 different cell lines (LNCaP, PC3, HCT 115, COLO 205, and MCF7) along with pRL-TK Renilla luciferase plasmid for 24 h. Dual luciferase assays were conducted. The results were normalized against the internal Renilla control for each transfection. The luciferase activity of the control region was defined as 1. Relative luciferase activity values are presented as mean±SD of triplicate transfections.

Mentions: We cloned approximately 1.5-kb DNA fragments, centered on AcPs from LNCaP, HCT116 or MCF7 cells, upstream of a luciferase reporter gene driven by the thymidine kinase (TK) minimal promoter. Enhancer activities of the fragments were determined by transient transfection and luciferase assays in LNCaP & PC3 (prostate cancer cells), HCT116 & COLO 205 (colorectal cancer cells) and MCF7 (breast cancer cells) (Figure 4). AcP6 (in the breast cancer risk region) and AcP10 (in prostate cancer/colorectal cancer risk region 3) had the most pronounced enhancer activities, whereas AcPs12 – 15 (in prostate cancer risk region 1) had activities that were lower, but clear compared to the negative control and several other AcPs. Interestingly, these active enhancers also displayed unmistakable H3K4me1 and H3K4me3 marks. The results suggest that some of the active chromatin foci we identified (Figure 3, right inset) have intrinsic enhancer activities within cellular contexts. This concept was further supported in a parallel study, in colorectal cells, which demonstrated that region 3, encompassing AcP10 and harboring SNP rs6983267, bound transcription factor T-cell factor 4 (TCF4) in an allele specific manner [17]. In the present study we did not study this region but rather analyzed region 1 further in prostate cancer cells.


Functional enhancers at the gene-poor 8q24 cancer-linked locus.

Jia L, Landan G, Pomerantz M, Jaschek R, Herman P, Reich D, Yan C, Khalid O, Kantoff P, Oh W, Manak JR, Berman BP, Henderson BE, Frenkel B, Haiman CA, Freedman M, Tanay A, Coetzee GA - PLoS Genet. (2009)

Constitutive enhancer activity of AcH3 peak sequences at 8q24.The DNA sequence containing each of the 15 identified AcH3 sites or a control sequence from the neighboring unacetylated region was inserted upstream of TK-luciferase reporter vector. The constructs were transfected into 5 different cell lines (LNCaP, PC3, HCT 115, COLO 205, and MCF7) along with pRL-TK Renilla luciferase plasmid for 24 h. Dual luciferase assays were conducted. The results were normalized against the internal Renilla control for each transfection. The luciferase activity of the control region was defined as 1. Relative luciferase activity values are presented as mean±SD of triplicate transfections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2717370&req=5

pgen-1000597-g004: Constitutive enhancer activity of AcH3 peak sequences at 8q24.The DNA sequence containing each of the 15 identified AcH3 sites or a control sequence from the neighboring unacetylated region was inserted upstream of TK-luciferase reporter vector. The constructs were transfected into 5 different cell lines (LNCaP, PC3, HCT 115, COLO 205, and MCF7) along with pRL-TK Renilla luciferase plasmid for 24 h. Dual luciferase assays were conducted. The results were normalized against the internal Renilla control for each transfection. The luciferase activity of the control region was defined as 1. Relative luciferase activity values are presented as mean±SD of triplicate transfections.
Mentions: We cloned approximately 1.5-kb DNA fragments, centered on AcPs from LNCaP, HCT116 or MCF7 cells, upstream of a luciferase reporter gene driven by the thymidine kinase (TK) minimal promoter. Enhancer activities of the fragments were determined by transient transfection and luciferase assays in LNCaP & PC3 (prostate cancer cells), HCT116 & COLO 205 (colorectal cancer cells) and MCF7 (breast cancer cells) (Figure 4). AcP6 (in the breast cancer risk region) and AcP10 (in prostate cancer/colorectal cancer risk region 3) had the most pronounced enhancer activities, whereas AcPs12 – 15 (in prostate cancer risk region 1) had activities that were lower, but clear compared to the negative control and several other AcPs. Interestingly, these active enhancers also displayed unmistakable H3K4me1 and H3K4me3 marks. The results suggest that some of the active chromatin foci we identified (Figure 3, right inset) have intrinsic enhancer activities within cellular contexts. This concept was further supported in a parallel study, in colorectal cells, which demonstrated that region 3, encompassing AcP10 and harboring SNP rs6983267, bound transcription factor T-cell factor 4 (TCF4) in an allele specific manner [17]. In the present study we did not study this region but rather analyzed region 1 further in prostate cancer cells.

Bottom Line: Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR).Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness.The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

View Article: PubMed Central - PubMed

Affiliation: USC/Norris Cancer Center, Department of Preventive Medicine, University of Southern California, Los Angeles, CA, USA.

ABSTRACT
Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

Show MeSH
Related in: MedlinePlus