Limits...
Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer.

Nanni S, Aiello A, Re A, Guffanti A, Benvenuti V, Colussi C, Castro-Vega LJ, Felsani A, Londono-Vallejo A, Capogrossi MC, Bacchetti S, Gaetano C, Pontecorvi A, Farsetti A - PLoS ONE (2013)

Bottom Line: To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells.The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS.E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, National Cancer Institute Regina Elena, Rome, Italy.

ABSTRACT
In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5' domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

Show MeSH

Related in: MedlinePlus

Global overview of eNOS-recruitment by ChIP sequencing and genome-wide changes mediated by estradiol.A) Venn diagram of regions displaying eNOS-recruitment in the absence (NT) or presence of estradiol (E2). Number of discrete genomic eNOS-recruitment peaks identified by ChIP-Seq in C27IM_NT and C27IM_E2 (left), LNCaP_NT and LNCaP_E2 (right) using MACS analysis (FDR<0,1 and P value p<1e-5). Overlap between peaks in NT and E2condition was determined using threshold of 1 nt. B) Length distribution of eNOS peaks in C27 IM_NT, C27 IM_ E2 (left) and LNCaP NT, LNCaP E2 (right). Data are presented as superimposed box plots of peak lenghts for E2 treated (black) or untreated (gray). Black line is the E2 peaks mean length, gray line is the NT peaks mean length. p<2.2e-16 NT vs E2. C) Pie chart of eNOS-peaks distribution in intra/extragenic regions (left) or of distance from TSS (right). Numbers in percentage represent min-max values in each category. D) Venn diagram of MACS-peaks in C27IM_E2 and LNCaP_E2. Overlap between eNOS peaks was determined using threshold of 1 nt. E) Validation by quantitative PCR of ChIP-Seq eNOS-peaks. eNOS binding was monitored in 9 genomic regions, specificity was assessed in an “empty” region (region without eNOS peaks) of chromosome 5. Data represent mean+/−SEM of 3 independent experiments. *p<0,05 eNOS_E2 vs eNOS_NT.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643940&req=5

pone-0062522-g001: Global overview of eNOS-recruitment by ChIP sequencing and genome-wide changes mediated by estradiol.A) Venn diagram of regions displaying eNOS-recruitment in the absence (NT) or presence of estradiol (E2). Number of discrete genomic eNOS-recruitment peaks identified by ChIP-Seq in C27IM_NT and C27IM_E2 (left), LNCaP_NT and LNCaP_E2 (right) using MACS analysis (FDR<0,1 and P value p<1e-5). Overlap between peaks in NT and E2condition was determined using threshold of 1 nt. B) Length distribution of eNOS peaks in C27 IM_NT, C27 IM_ E2 (left) and LNCaP NT, LNCaP E2 (right). Data are presented as superimposed box plots of peak lenghts for E2 treated (black) or untreated (gray). Black line is the E2 peaks mean length, gray line is the NT peaks mean length. p<2.2e-16 NT vs E2. C) Pie chart of eNOS-peaks distribution in intra/extragenic regions (left) or of distance from TSS (right). Numbers in percentage represent min-max values in each category. D) Venn diagram of MACS-peaks in C27IM_E2 and LNCaP_E2. Overlap between eNOS peaks was determined using threshold of 1 nt. E) Validation by quantitative PCR of ChIP-Seq eNOS-peaks. eNOS binding was monitored in 9 genomic regions, specificity was assessed in an “empty” region (region without eNOS peaks) of chromosome 5. Data represent mean+/−SEM of 3 independent experiments. *p<0,05 eNOS_E2 vs eNOS_NT.

Mentions: We identified by MACS peak call analysis in C27IM and LNCaP cells, respectively 12,034 and 2,344 eNOS-associated peaks before E2 treatment, and 57,802 and 34,560 thereafter (Table S2). Of note, the removal of open chromatin regions known to generate false positives in ChIP-seq experiments (the so-called “ultra-high signal artifact regions”) left substantially unchanged the results in terms of peak number: 11,694 and 2,333 in untreated C27IM and LNCaP cells and 57,616 or 34,451 in C27IM and LNCaP cells upon E2 treatment (Table S2 and Figure 1A) (see Methods and [32].


Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer.

Nanni S, Aiello A, Re A, Guffanti A, Benvenuti V, Colussi C, Castro-Vega LJ, Felsani A, Londono-Vallejo A, Capogrossi MC, Bacchetti S, Gaetano C, Pontecorvi A, Farsetti A - PLoS ONE (2013)

Global overview of eNOS-recruitment by ChIP sequencing and genome-wide changes mediated by estradiol.A) Venn diagram of regions displaying eNOS-recruitment in the absence (NT) or presence of estradiol (E2). Number of discrete genomic eNOS-recruitment peaks identified by ChIP-Seq in C27IM_NT and C27IM_E2 (left), LNCaP_NT and LNCaP_E2 (right) using MACS analysis (FDR<0,1 and P value p<1e-5). Overlap between peaks in NT and E2condition was determined using threshold of 1 nt. B) Length distribution of eNOS peaks in C27 IM_NT, C27 IM_ E2 (left) and LNCaP NT, LNCaP E2 (right). Data are presented as superimposed box plots of peak lenghts for E2 treated (black) or untreated (gray). Black line is the E2 peaks mean length, gray line is the NT peaks mean length. p<2.2e-16 NT vs E2. C) Pie chart of eNOS-peaks distribution in intra/extragenic regions (left) or of distance from TSS (right). Numbers in percentage represent min-max values in each category. D) Venn diagram of MACS-peaks in C27IM_E2 and LNCaP_E2. Overlap between eNOS peaks was determined using threshold of 1 nt. E) Validation by quantitative PCR of ChIP-Seq eNOS-peaks. eNOS binding was monitored in 9 genomic regions, specificity was assessed in an “empty” region (region without eNOS peaks) of chromosome 5. Data represent mean+/−SEM of 3 independent experiments. *p<0,05 eNOS_E2 vs eNOS_NT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643940&req=5

pone-0062522-g001: Global overview of eNOS-recruitment by ChIP sequencing and genome-wide changes mediated by estradiol.A) Venn diagram of regions displaying eNOS-recruitment in the absence (NT) or presence of estradiol (E2). Number of discrete genomic eNOS-recruitment peaks identified by ChIP-Seq in C27IM_NT and C27IM_E2 (left), LNCaP_NT and LNCaP_E2 (right) using MACS analysis (FDR<0,1 and P value p<1e-5). Overlap between peaks in NT and E2condition was determined using threshold of 1 nt. B) Length distribution of eNOS peaks in C27 IM_NT, C27 IM_ E2 (left) and LNCaP NT, LNCaP E2 (right). Data are presented as superimposed box plots of peak lenghts for E2 treated (black) or untreated (gray). Black line is the E2 peaks mean length, gray line is the NT peaks mean length. p<2.2e-16 NT vs E2. C) Pie chart of eNOS-peaks distribution in intra/extragenic regions (left) or of distance from TSS (right). Numbers in percentage represent min-max values in each category. D) Venn diagram of MACS-peaks in C27IM_E2 and LNCaP_E2. Overlap between eNOS peaks was determined using threshold of 1 nt. E) Validation by quantitative PCR of ChIP-Seq eNOS-peaks. eNOS binding was monitored in 9 genomic regions, specificity was assessed in an “empty” region (region without eNOS peaks) of chromosome 5. Data represent mean+/−SEM of 3 independent experiments. *p<0,05 eNOS_E2 vs eNOS_NT.
Mentions: We identified by MACS peak call analysis in C27IM and LNCaP cells, respectively 12,034 and 2,344 eNOS-associated peaks before E2 treatment, and 57,802 and 34,560 thereafter (Table S2). Of note, the removal of open chromatin regions known to generate false positives in ChIP-seq experiments (the so-called “ultra-high signal artifact regions”) left substantially unchanged the results in terms of peak number: 11,694 and 2,333 in untreated C27IM and LNCaP cells and 57,616 or 34,451 in C27IM and LNCaP cells upon E2 treatment (Table S2 and Figure 1A) (see Methods and [32].

Bottom Line: To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells.The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS.E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, National Cancer Institute Regina Elena, Rome, Italy.

ABSTRACT
In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5' domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

Show MeSH
Related in: MedlinePlus