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Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells.

Boo K, Bhin J, Jeon Y, Kim J, Shin HJ, Park JE, Kim K, Kim CR, Jang H, Kim IH, Kim VN, Hwang D, Lee H, Baek SH - Nat Commun (2015)

Bottom Line: The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states.Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells.Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Creative Research Initiative Center for Chromatin Dynamics, School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.

ABSTRACT
The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

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Related in: MedlinePlus

Identification of target genes regulated by Pontin in mouse ES cells.(a) Up- and downregulated genes in Pontin-depleted ES cells in comparison with Pontinf/f; CreER ES cells at 3 days (OHT 3D) or 4 days (OHT 4D) versus 0 day post-OHT treatment (-OHT). Hierarchical clustering identified six clusters of the up- (C1-C3) and downregulated (C4-C6) genes. Numbers of DEGs in the clusters are denoted in parenthesis. The colour bar represents the gradient of log2-fold-changes in each comparison. (b) Gene Ontology Biological Processes (GOBPs) represented by the up- and downregulated genes by Pontin depletion (dark bars) and by both Pontin depletion and Oct4 depletion (light bars). The bars for GOBPs represent the enrichment scores, −log10(p), where p is P value that the GOBPs are enriched. The red line denotes the cutoff, P=0.1. (c) mRNA-sequencing reads of two upregulated genes, Timp2 and Tpm1, and two downregulated genes, Rif1 and Ptch1, in Pontin-depleted ES cells. Red and blue bars (y axis) along the genomic coordinate (x axis) represent read coverages for individual bases of the genes measured at OHT 4D from Pontin-depleted and WT ES cells, respectively. Each bar graph shows the normalized read counts of the corresponding gene in WT and Pontin-depleted ES cells. (d) Quantitative RT-PCR analysis of up- and downregulated genes in Pontin-depleted ES cells. The mRNA quantity was normalized by using primers to detect Gapdh. Error bars represent mean±s.d.; *P<0.05. (e) Comparison of ES cell expression profiles on gene depletion (KO) or knockdown (KD) of the indicated factors using hierarchical clustering of Pearson correlation coefficients of log2-fold-changes in KO or KD ES cells, compared with WT ES cells. Changes of gene expression in Dnmt1-depleted ES cells were used as the negative control. (f) Comparisons of binding enrichments of Pontin with those of Oct4, Sox2, Nanog, Tip60 and p400 from ChIP-sequencing analysis. X axis indicates the sorted genes by Pontin enrichment ratios, log2(Pontin-ChIP/GFP). Left y axis indicates the mean enrichment ratios of Pontin in individual bins of the sorted genes (500 genes/bin), and right y axis indicates the mean enrichment ratios of the other factors for the genes in the individual bins defined by Pontin.
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f2: Identification of target genes regulated by Pontin in mouse ES cells.(a) Up- and downregulated genes in Pontin-depleted ES cells in comparison with Pontinf/f; CreER ES cells at 3 days (OHT 3D) or 4 days (OHT 4D) versus 0 day post-OHT treatment (-OHT). Hierarchical clustering identified six clusters of the up- (C1-C3) and downregulated (C4-C6) genes. Numbers of DEGs in the clusters are denoted in parenthesis. The colour bar represents the gradient of log2-fold-changes in each comparison. (b) Gene Ontology Biological Processes (GOBPs) represented by the up- and downregulated genes by Pontin depletion (dark bars) and by both Pontin depletion and Oct4 depletion (light bars). The bars for GOBPs represent the enrichment scores, −log10(p), where p is P value that the GOBPs are enriched. The red line denotes the cutoff, P=0.1. (c) mRNA-sequencing reads of two upregulated genes, Timp2 and Tpm1, and two downregulated genes, Rif1 and Ptch1, in Pontin-depleted ES cells. Red and blue bars (y axis) along the genomic coordinate (x axis) represent read coverages for individual bases of the genes measured at OHT 4D from Pontin-depleted and WT ES cells, respectively. Each bar graph shows the normalized read counts of the corresponding gene in WT and Pontin-depleted ES cells. (d) Quantitative RT-PCR analysis of up- and downregulated genes in Pontin-depleted ES cells. The mRNA quantity was normalized by using primers to detect Gapdh. Error bars represent mean±s.d.; *P<0.05. (e) Comparison of ES cell expression profiles on gene depletion (KO) or knockdown (KD) of the indicated factors using hierarchical clustering of Pearson correlation coefficients of log2-fold-changes in KO or KD ES cells, compared with WT ES cells. Changes of gene expression in Dnmt1-depleted ES cells were used as the negative control. (f) Comparisons of binding enrichments of Pontin with those of Oct4, Sox2, Nanog, Tip60 and p400 from ChIP-sequencing analysis. X axis indicates the sorted genes by Pontin enrichment ratios, log2(Pontin-ChIP/GFP). Left y axis indicates the mean enrichment ratios of Pontin in individual bins of the sorted genes (500 genes/bin), and right y axis indicates the mean enrichment ratios of the other factors for the genes in the individual bins defined by Pontin.

Mentions: To gain insights into the underlying mechanisms by which Pontin contributes to the ES cell maintenance, we performed genome-wide mRNA-sequencing analysis of Pontinf/f; CreER ES cells at 0, 3 or 4 days post-treatment with OHT (Fig. 2a). On average, 55.4 million reads were obtained in individual samples and aligned to the mouse genome, resulting in 5.3 Giga bps of mapped sequences, which corresponds to 52.2-fold coverage of the annotated mouse transcriptome (Supplementary Data 1). To explore the downstream target genes of Pontin, we compared gene expression in Pontin-depleted ES cells relative to that of WT and identified differentially expressed genes (DEGs) that consist of 1,205 upregulated and 1,678 downregulated genes in Pontin-depleted ES cells compared with WT (Fig. 2a). The cellular processes affected by Pontin were then examined by performing functional enrichment analysis of the DEGs using DAVID software40 (Fig. 2b and Supplementary Data 2). This analysis indicates that the majority of upregulated genes in Pontin-depleted ES cells are mainly related to differentiation and embryonic developmental processes, whereas downregulated genes are involved in cell cycle and metabolism, which are essential for ES cell maintenance (Fig. 2b). Consistent with this observation, the genes related to development and differentiation (that is, Timp2 and Tpm1) are upregulated in Pontin-depleted ES cells, whereas the genes involved in stem cell maintenance and metabolism (that is, Rif1 and Ptch1) are downregulated as evidenced by mRNA sequencing (Fig. 2c) and quantitative RT-PCR analyses (Fig. 2d).


Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells.

Boo K, Bhin J, Jeon Y, Kim J, Shin HJ, Park JE, Kim K, Kim CR, Jang H, Kim IH, Kim VN, Hwang D, Lee H, Baek SH - Nat Commun (2015)

Identification of target genes regulated by Pontin in mouse ES cells.(a) Up- and downregulated genes in Pontin-depleted ES cells in comparison with Pontinf/f; CreER ES cells at 3 days (OHT 3D) or 4 days (OHT 4D) versus 0 day post-OHT treatment (-OHT). Hierarchical clustering identified six clusters of the up- (C1-C3) and downregulated (C4-C6) genes. Numbers of DEGs in the clusters are denoted in parenthesis. The colour bar represents the gradient of log2-fold-changes in each comparison. (b) Gene Ontology Biological Processes (GOBPs) represented by the up- and downregulated genes by Pontin depletion (dark bars) and by both Pontin depletion and Oct4 depletion (light bars). The bars for GOBPs represent the enrichment scores, −log10(p), where p is P value that the GOBPs are enriched. The red line denotes the cutoff, P=0.1. (c) mRNA-sequencing reads of two upregulated genes, Timp2 and Tpm1, and two downregulated genes, Rif1 and Ptch1, in Pontin-depleted ES cells. Red and blue bars (y axis) along the genomic coordinate (x axis) represent read coverages for individual bases of the genes measured at OHT 4D from Pontin-depleted and WT ES cells, respectively. Each bar graph shows the normalized read counts of the corresponding gene in WT and Pontin-depleted ES cells. (d) Quantitative RT-PCR analysis of up- and downregulated genes in Pontin-depleted ES cells. The mRNA quantity was normalized by using primers to detect Gapdh. Error bars represent mean±s.d.; *P<0.05. (e) Comparison of ES cell expression profiles on gene depletion (KO) or knockdown (KD) of the indicated factors using hierarchical clustering of Pearson correlation coefficients of log2-fold-changes in KO or KD ES cells, compared with WT ES cells. Changes of gene expression in Dnmt1-depleted ES cells were used as the negative control. (f) Comparisons of binding enrichments of Pontin with those of Oct4, Sox2, Nanog, Tip60 and p400 from ChIP-sequencing analysis. X axis indicates the sorted genes by Pontin enrichment ratios, log2(Pontin-ChIP/GFP). Left y axis indicates the mean enrichment ratios of Pontin in individual bins of the sorted genes (500 genes/bin), and right y axis indicates the mean enrichment ratios of the other factors for the genes in the individual bins defined by Pontin.
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f2: Identification of target genes regulated by Pontin in mouse ES cells.(a) Up- and downregulated genes in Pontin-depleted ES cells in comparison with Pontinf/f; CreER ES cells at 3 days (OHT 3D) or 4 days (OHT 4D) versus 0 day post-OHT treatment (-OHT). Hierarchical clustering identified six clusters of the up- (C1-C3) and downregulated (C4-C6) genes. Numbers of DEGs in the clusters are denoted in parenthesis. The colour bar represents the gradient of log2-fold-changes in each comparison. (b) Gene Ontology Biological Processes (GOBPs) represented by the up- and downregulated genes by Pontin depletion (dark bars) and by both Pontin depletion and Oct4 depletion (light bars). The bars for GOBPs represent the enrichment scores, −log10(p), where p is P value that the GOBPs are enriched. The red line denotes the cutoff, P=0.1. (c) mRNA-sequencing reads of two upregulated genes, Timp2 and Tpm1, and two downregulated genes, Rif1 and Ptch1, in Pontin-depleted ES cells. Red and blue bars (y axis) along the genomic coordinate (x axis) represent read coverages for individual bases of the genes measured at OHT 4D from Pontin-depleted and WT ES cells, respectively. Each bar graph shows the normalized read counts of the corresponding gene in WT and Pontin-depleted ES cells. (d) Quantitative RT-PCR analysis of up- and downregulated genes in Pontin-depleted ES cells. The mRNA quantity was normalized by using primers to detect Gapdh. Error bars represent mean±s.d.; *P<0.05. (e) Comparison of ES cell expression profiles on gene depletion (KO) or knockdown (KD) of the indicated factors using hierarchical clustering of Pearson correlation coefficients of log2-fold-changes in KO or KD ES cells, compared with WT ES cells. Changes of gene expression in Dnmt1-depleted ES cells were used as the negative control. (f) Comparisons of binding enrichments of Pontin with those of Oct4, Sox2, Nanog, Tip60 and p400 from ChIP-sequencing analysis. X axis indicates the sorted genes by Pontin enrichment ratios, log2(Pontin-ChIP/GFP). Left y axis indicates the mean enrichment ratios of Pontin in individual bins of the sorted genes (500 genes/bin), and right y axis indicates the mean enrichment ratios of the other factors for the genes in the individual bins defined by Pontin.
Mentions: To gain insights into the underlying mechanisms by which Pontin contributes to the ES cell maintenance, we performed genome-wide mRNA-sequencing analysis of Pontinf/f; CreER ES cells at 0, 3 or 4 days post-treatment with OHT (Fig. 2a). On average, 55.4 million reads were obtained in individual samples and aligned to the mouse genome, resulting in 5.3 Giga bps of mapped sequences, which corresponds to 52.2-fold coverage of the annotated mouse transcriptome (Supplementary Data 1). To explore the downstream target genes of Pontin, we compared gene expression in Pontin-depleted ES cells relative to that of WT and identified differentially expressed genes (DEGs) that consist of 1,205 upregulated and 1,678 downregulated genes in Pontin-depleted ES cells compared with WT (Fig. 2a). The cellular processes affected by Pontin were then examined by performing functional enrichment analysis of the DEGs using DAVID software40 (Fig. 2b and Supplementary Data 2). This analysis indicates that the majority of upregulated genes in Pontin-depleted ES cells are mainly related to differentiation and embryonic developmental processes, whereas downregulated genes are involved in cell cycle and metabolism, which are essential for ES cell maintenance (Fig. 2b). Consistent with this observation, the genes related to development and differentiation (that is, Timp2 and Tpm1) are upregulated in Pontin-depleted ES cells, whereas the genes involved in stem cell maintenance and metabolism (that is, Rif1 and Ptch1) are downregulated as evidenced by mRNA sequencing (Fig. 2c) and quantitative RT-PCR analyses (Fig. 2d).

Bottom Line: The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states.Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells.Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Creative Research Initiative Center for Chromatin Dynamics, School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.

ABSTRACT
The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

Show MeSH
Related in: MedlinePlus