Project description:It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression. For expression profiling of cells in NME2-induced conditions, A549 cells were transfected with pcDNA-NME2-MYC or pcDNA-MYC (control). RNA was isolated from the cells 48h after transfection using the trizol method (Sigma) as per manufacturer’s protocol. Total RNA was processed to hybridize to Illumina Human HT-12 v4 Expression BeadChip as per manufacturer’s instructions. Three biological replicates were averaged and data was analyzed using BeadStudio (P <0.05 of fold change).

Project description:It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression.

Project description:It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression. For expression profiling of cells in NME2-induced conditions, A549 cells were transfected with pcDNA-NME2-MYC or pcDNA-MYC (control). RNA was isolated from the cells 48h after transfection using the trizol method (Sigma) as per manufacturerM-bM-^@M-^Ys protocol. Total RNA was processed to hybridize to Illumina Human HT-12 v4 Expression BeadChip as per manufacturerM-bM-^@M-^Ys instructions. Three biological replicates were averaged and data was analyzed using BeadStudio (P <0.05 of fold change).

Project description:BackgroundNm23-H1 gene has been found to be an inhibitor of tumor metastasis in lung cancer. MicroRNAs (miRNAs) play key roles in tumor metastasis through multiple signaling pathways. This study explored whether the nm23-H1 gene could inhibit invasion and metastasis of lung cancer cells by regulating miRNA-660-5p targets.MethodsQuantitative real-time PCR (qRT-PCR) and western blots were used to measure the expression of nm23-H1 and miR-660-5p of various human lung cancer cell lines. Cell counting kit-8 (CCK-8), wound-healing and transwell assay were carried out to assess cell proliferation, migration and invasion of each cell line. Xenograft were applied to determine in vivo effects of miR-660-5p among nude mice. Luciferase assay and western blot were performed to determine the target gene of miR-660-5p.ResultsWe found that high expression of nm23-H1 correlated with decreased miRNA-660-5p expression. Inhibiting miR-660-5p suppressed lung cancer cells progression significantly in vitro, whereas overexpression of miR-660-5p facilitated tumor growth and bone metastasis in vivo. In addition, as the potential target gene of miR-660-5p, SMARCA5 overexpression in vitro suppressed tumor progression and osteolytic metastasis associated RANKL signaling, which is congruent with the effect of nm23-H1 on the lung cancer cells.ConclusionNm23-H1 inhibits tumor progression and bone-specific metastasis of lung cancer by regulating miR-660-5p/SMARCA5/RANKL axis, which indicates the related genes may serve as potential targets for the treatment of human lung cancer.Key pointsSignificant findings of the study High expression of nm23-H1 correlated with decreased miRNA-660-5p expression. Further, downregulation of miR-660-5p significantly suppressed the tumor progression and bone-specific metastasis of lung cancer cells. What this study adds This is the first study to show an inverse association between nm23-H1 and miR-660-5p, and confirm that nm23-H1 inhibits tumor progression and bone-specific metastasis of lung cancer by regulating miR-660-5p/SMARCA5/RANKL axis.

Dataset Information

Expression profile of lung adenocarcinoma, A549 cells following targeted depletion of non metastatic 2 (NME2/NM23 H2)

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