Limits...
Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

Savic D, Ramaker RC, Roberts BS, Dean EC, Burwell TC, Meadows SK, Cooper SJ, Garabedian MJ, Gertz J, Myers RM - Genome Med (2016)

Bottom Line: PPARG generated a rapid and short-term response while maintaining a gene activator role.By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points.Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome.

View Article: PubMed Central - PubMed

Affiliation: HudsonAlpha Institute for Biotechnology, Huntsville, AL, 35806, USA.

ABSTRACT

Background: The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking.

Methods: To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns.

Results: Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points.

Conclusions: Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

No MeSH data available.


Related in: MedlinePlus

LXR repression is consistent with RNAP2 promoter-proximal pausing. a Smooth scatter plot comparing RNA-derived gene expression levels after 48-h GW3965 treatment (y-axis, reads per kilobase of transcript per million mapped reads (RPKM)) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of GW3965 (48 h) normalized to control culture conditions (0 h). Data are presented for all GW3965 responsive genes (adjusted p < 0.01, fold change cutoff of ±2). b Smooth scatter plot comparing RNA-derived gene expression levels after 48 h of rosiglitazone stimulation (y-axis, RPKM) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of rosiglitazone (48 h) normalized to control culture conditions (0 h). Data are presented for all rosiglitazone-responsive genes (adjusted p < 0.01, fold change cutoff of ±2). c Histogram tabulating the read depth ratios (x-axis) of RNAP2 enrichment sites co-occupied by LXRA and RNAP2 (top panel) and sites co-occupied by LXRB and RNAP2 (bottom panel). Co-occupied sites are divided based on promoter location (Non-Promoter and Promoter). Negative values highlight stronger enrichment under control culture conditions (0 h) while positive values denote stronger enrichment after 48 hours of GW3965 treatment. d Histogram tabulating the read depth ratios (x-axis) of H3K36me3 enrichment in gene bodies for genes down-regulated after 48 h of GW3965 treatment. Negative values highlight stronger enrichment at baseline (0 h) while positive values denote stronger enrichment after 48 h of GW3965 treatment. e Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRA binding events within 10 kb (<10 kb) from promoters, as well as plots for genes with no evidence of nearby LXRA occupancy (>100 kb). f Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRB binding events within 10 kb (<10 kb) of promoters, as well as plots for genes with no evidence of nearby LXRB occupancy (>100 kb)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940857&req=5

Fig6: LXR repression is consistent with RNAP2 promoter-proximal pausing. a Smooth scatter plot comparing RNA-derived gene expression levels after 48-h GW3965 treatment (y-axis, reads per kilobase of transcript per million mapped reads (RPKM)) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of GW3965 (48 h) normalized to control culture conditions (0 h). Data are presented for all GW3965 responsive genes (adjusted p < 0.01, fold change cutoff of ±2). b Smooth scatter plot comparing RNA-derived gene expression levels after 48 h of rosiglitazone stimulation (y-axis, RPKM) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of rosiglitazone (48 h) normalized to control culture conditions (0 h). Data are presented for all rosiglitazone-responsive genes (adjusted p < 0.01, fold change cutoff of ±2). c Histogram tabulating the read depth ratios (x-axis) of RNAP2 enrichment sites co-occupied by LXRA and RNAP2 (top panel) and sites co-occupied by LXRB and RNAP2 (bottom panel). Co-occupied sites are divided based on promoter location (Non-Promoter and Promoter). Negative values highlight stronger enrichment under control culture conditions (0 h) while positive values denote stronger enrichment after 48 hours of GW3965 treatment. d Histogram tabulating the read depth ratios (x-axis) of H3K36me3 enrichment in gene bodies for genes down-regulated after 48 h of GW3965 treatment. Negative values highlight stronger enrichment at baseline (0 h) while positive values denote stronger enrichment after 48 h of GW3965 treatment. e Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRA binding events within 10 kb (<10 kb) from promoters, as well as plots for genes with no evidence of nearby LXRA occupancy (>100 kb). f Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRB binding events within 10 kb (<10 kb) of promoters, as well as plots for genes with no evidence of nearby LXRB occupancy (>100 kb)

Mentions: To infer a potential mechanism for the late, LXR-mediated gene repression described above, we compared RNAP2 binding profiles to RNA-seq results (adjusted p < 0.01, fold change cutoff ±2). We first used our RNAP2 ChIP-seq data to determine the predictive value of RNAP2 normalized read depth at promoters as a measurement for global gene expression (Additional file 1: Figure S27). For all drug treatments, we obtained sufficient correlations (rho > 0.72). Having shown the utility of promoter RNAP2 occupancy, we next assessed RNAP2 read depth at promoters of differentially regulated genes (Fig. 6a, b; Additional file 1: Figures S28–S31). During the early response (Additional file 1: Figure S28), both datasets were well correlated; the vast majority of up-regulated genes exhibited stronger RNAP2 promoter enrichment compared with control culture conditions, while down-regulated genes had reduced RNAP2 read depth at their promoters. However, pronounced differences were observed between GW3965 and rosiglitazone treatments at late time points (Fig. 6a, b). Although 81 % of activated GW3965 genes displayed stronger RNAP2 promoter enrichment, paradoxically, only 34 % of repressed genes showed a decrease in RNAP2 promoter occupancy. This effect was not observed for rosiglitazone, where 81 % of up-regulated and 85 % of down-regulated genes maintained correlated changes with RNAP2 promoter occupancy. We further validated this deviation when we used lower fold change cutoffs (Additional file 1: Figures S29 and S30), while swapping binding sites and gene expression datasets between GW3965 and rosiglitazone treatments generated random, overlapping distributions between up- and down-regulated genes (Additional file 1: Figure S31).Fig. 6


Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

Savic D, Ramaker RC, Roberts BS, Dean EC, Burwell TC, Meadows SK, Cooper SJ, Garabedian MJ, Gertz J, Myers RM - Genome Med (2016)

LXR repression is consistent with RNAP2 promoter-proximal pausing. a Smooth scatter plot comparing RNA-derived gene expression levels after 48-h GW3965 treatment (y-axis, reads per kilobase of transcript per million mapped reads (RPKM)) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of GW3965 (48 h) normalized to control culture conditions (0 h). Data are presented for all GW3965 responsive genes (adjusted p < 0.01, fold change cutoff of ±2). b Smooth scatter plot comparing RNA-derived gene expression levels after 48 h of rosiglitazone stimulation (y-axis, RPKM) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of rosiglitazone (48 h) normalized to control culture conditions (0 h). Data are presented for all rosiglitazone-responsive genes (adjusted p < 0.01, fold change cutoff of ±2). c Histogram tabulating the read depth ratios (x-axis) of RNAP2 enrichment sites co-occupied by LXRA and RNAP2 (top panel) and sites co-occupied by LXRB and RNAP2 (bottom panel). Co-occupied sites are divided based on promoter location (Non-Promoter and Promoter). Negative values highlight stronger enrichment under control culture conditions (0 h) while positive values denote stronger enrichment after 48 hours of GW3965 treatment. d Histogram tabulating the read depth ratios (x-axis) of H3K36me3 enrichment in gene bodies for genes down-regulated after 48 h of GW3965 treatment. Negative values highlight stronger enrichment at baseline (0 h) while positive values denote stronger enrichment after 48 h of GW3965 treatment. e Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRA binding events within 10 kb (<10 kb) from promoters, as well as plots for genes with no evidence of nearby LXRA occupancy (>100 kb). f Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRB binding events within 10 kb (<10 kb) of promoters, as well as plots for genes with no evidence of nearby LXRB occupancy (>100 kb)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940857&req=5

Fig6: LXR repression is consistent with RNAP2 promoter-proximal pausing. a Smooth scatter plot comparing RNA-derived gene expression levels after 48-h GW3965 treatment (y-axis, reads per kilobase of transcript per million mapped reads (RPKM)) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of GW3965 (48 h) normalized to control culture conditions (0 h). Data are presented for all GW3965 responsive genes (adjusted p < 0.01, fold change cutoff of ±2). b Smooth scatter plot comparing RNA-derived gene expression levels after 48 h of rosiglitazone stimulation (y-axis, RPKM) with sequencing read depth ratios of ChIP-derived RNAP2 promoter occupancy (x-axis). Read depth at promoters assesses the enrichment after 48 h of rosiglitazone (48 h) normalized to control culture conditions (0 h). Data are presented for all rosiglitazone-responsive genes (adjusted p < 0.01, fold change cutoff of ±2). c Histogram tabulating the read depth ratios (x-axis) of RNAP2 enrichment sites co-occupied by LXRA and RNAP2 (top panel) and sites co-occupied by LXRB and RNAP2 (bottom panel). Co-occupied sites are divided based on promoter location (Non-Promoter and Promoter). Negative values highlight stronger enrichment under control culture conditions (0 h) while positive values denote stronger enrichment after 48 hours of GW3965 treatment. d Histogram tabulating the read depth ratios (x-axis) of H3K36me3 enrichment in gene bodies for genes down-regulated after 48 h of GW3965 treatment. Negative values highlight stronger enrichment at baseline (0 h) while positive values denote stronger enrichment after 48 h of GW3965 treatment. e Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRA binding events within 10 kb (<10 kb) from promoters, as well as plots for genes with no evidence of nearby LXRA occupancy (>100 kb). f Violin plot of changes in H3K36me3 read depth ratio in gene bodies of repressed genes after 48 h of GW3965 treatment. Plots are displayed for genes that harbor LXRB binding events within 10 kb (<10 kb) of promoters, as well as plots for genes with no evidence of nearby LXRB occupancy (>100 kb)
Mentions: To infer a potential mechanism for the late, LXR-mediated gene repression described above, we compared RNAP2 binding profiles to RNA-seq results (adjusted p < 0.01, fold change cutoff ±2). We first used our RNAP2 ChIP-seq data to determine the predictive value of RNAP2 normalized read depth at promoters as a measurement for global gene expression (Additional file 1: Figure S27). For all drug treatments, we obtained sufficient correlations (rho > 0.72). Having shown the utility of promoter RNAP2 occupancy, we next assessed RNAP2 read depth at promoters of differentially regulated genes (Fig. 6a, b; Additional file 1: Figures S28–S31). During the early response (Additional file 1: Figure S28), both datasets were well correlated; the vast majority of up-regulated genes exhibited stronger RNAP2 promoter enrichment compared with control culture conditions, while down-regulated genes had reduced RNAP2 read depth at their promoters. However, pronounced differences were observed between GW3965 and rosiglitazone treatments at late time points (Fig. 6a, b). Although 81 % of activated GW3965 genes displayed stronger RNAP2 promoter enrichment, paradoxically, only 34 % of repressed genes showed a decrease in RNAP2 promoter occupancy. This effect was not observed for rosiglitazone, where 81 % of up-regulated and 85 % of down-regulated genes maintained correlated changes with RNAP2 promoter occupancy. We further validated this deviation when we used lower fold change cutoffs (Additional file 1: Figures S29 and S30), while swapping binding sites and gene expression datasets between GW3965 and rosiglitazone treatments generated random, overlapping distributions between up- and down-regulated genes (Additional file 1: Figure S31).Fig. 6

Bottom Line: PPARG generated a rapid and short-term response while maintaining a gene activator role.By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points.Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome.

View Article: PubMed Central - PubMed

Affiliation: HudsonAlpha Institute for Biotechnology, Huntsville, AL, 35806, USA.

ABSTRACT

Background: The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking.

Methods: To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns.

Results: Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points.

Conclusions: Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

No MeSH data available.


Related in: MedlinePlus