Limits...
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.

Show MeSH
Pontin is required for transcription of a subset of lincRNAs regulated by Oct4.(a) Proportions of the downregulated genes in Pontin-depleted ES cells out of those in Oct4-depleted ES cells. (b) Venn diagram of the downregulated lincRNAs in Oct4-depleted and Pontin-depleted ES cells. Sixteen lincRNAs are downregulated in both Pontin-depleted and Oct4-depleted ES cells. (c) List of 16 lincRNAs that are downregulated in both Oct4-depleted and Pontin-depleted ES cells. Asterisk (*) denotes the lincRNAs previously reported to function in repression of lineage specification process in ES cells. (d) Quantitative RT-PCR analysis of five lincRNAs involved in repression of differentiation processes among 16 lincRNAs that are downregulated in both Pontin-depleted and Oct4-depleted ES cells. The quantity of lincRNA was normalized by using primers to detect Gapdh. Values are expressed as mean±s.d. of three independent experiments. *P<0.05. (e) Luciferase assay was performed with reporters driven by linc1253 promoter possessing Oct4-binding elements (linc1253-1000) or deleted Oct4-binding region (linc1253-500). Luciferase activities were normalized by β-galactosidase activity. Values are expressed as mean±s.d. of three independent experiments. **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403444&req=5

f5: Pontin is required for transcription of a subset of lincRNAs regulated by Oct4.(a) Proportions of the downregulated genes in Pontin-depleted ES cells out of those in Oct4-depleted ES cells. (b) Venn diagram of the downregulated lincRNAs in Oct4-depleted and Pontin-depleted ES cells. Sixteen lincRNAs are downregulated in both Pontin-depleted and Oct4-depleted ES cells. (c) List of 16 lincRNAs that are downregulated in both Oct4-depleted and Pontin-depleted ES cells. Asterisk (*) denotes the lincRNAs previously reported to function in repression of lineage specification process in ES cells. (d) Quantitative RT-PCR analysis of five lincRNAs involved in repression of differentiation processes among 16 lincRNAs that are downregulated in both Pontin-depleted and Oct4-depleted ES cells. The quantity of lincRNA was normalized by using primers to detect Gapdh. Values are expressed as mean±s.d. of three independent experiments. *P<0.05. (e) Luciferase assay was performed with reporters driven by linc1253 promoter possessing Oct4-binding elements (linc1253-1000) or deleted Oct4-binding region (linc1253-500). Luciferase activities were normalized by β-galactosidase activity. Values are expressed as mean±s.d. of three independent experiments. **p<0.01.

Mentions: Chromatin signature mapping and global gene expression analysis with RNAi screening in ES cells showed that lincRNAs play a role in the maintenance of self-renewal and pluripotency. As a subset of lincRNAs has been shown to be directly regulated by Oct4, we tested the possibility that Pontin regulates expression of Oct4-dependent lincRNAs. Our analysis of Oct4 targets affected by Pontin revealed that lincRNAs are significantly affected by Pontin expression (Fig. 5a). Among the known 226 lincRNAs expressed in ES cells, 54 lincRNAs are downregulated in Oct4-depleted ES cells and 16 (30% in Fig. 5a, P=2.80 × 10−3 by Fisher's exact test) of the 54 lincRNAs are also downregulated in Pontin-depleted ES cells (Fig. 5b and Supplementary Data 4). Interestingly, five lincRNAs (linc1253, linc1356, linc1517, linc1562 and linc1602) that are co-regulated by both Pontin and Oct4 from our analysis (denoted with asterisk in Fig. 5c) have been shown to function as repressors of lineage differentiation programme in ES cells18. Moreover, the genes that are upregulated by knockdown of linc1253, linc1356 or linc1517 significantly overlapped with those that are upregulated by Pontin depletion (false discovery rate<0.1) (Supplementary Fig. 4a). These analyses support that Pontin and lincRNAs may collaborate to repress lineage specification programmes in ES cells. Quantitative RT-PCR analysis confirmed that lack of either Oct4 or Pontin resulted in downregulation of transcript levels of five lincRNAs that function to repress lineage differentiation programme (Fig. 5d).


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)

Pontin is required for transcription of a subset of lincRNAs regulated by Oct4.(a) Proportions of the downregulated genes in Pontin-depleted ES cells out of those in Oct4-depleted ES cells. (b) Venn diagram of the downregulated lincRNAs in Oct4-depleted and Pontin-depleted ES cells. Sixteen lincRNAs are downregulated in both Pontin-depleted and Oct4-depleted ES cells. (c) List of 16 lincRNAs that are downregulated in both Oct4-depleted and Pontin-depleted ES cells. Asterisk (*) denotes the lincRNAs previously reported to function in repression of lineage specification process in ES cells. (d) Quantitative RT-PCR analysis of five lincRNAs involved in repression of differentiation processes among 16 lincRNAs that are downregulated in both Pontin-depleted and Oct4-depleted ES cells. The quantity of lincRNA was normalized by using primers to detect Gapdh. Values are expressed as mean±s.d. of three independent experiments. *P<0.05. (e) Luciferase assay was performed with reporters driven by linc1253 promoter possessing Oct4-binding elements (linc1253-1000) or deleted Oct4-binding region (linc1253-500). Luciferase activities were normalized by β-galactosidase activity. Values are expressed as mean±s.d. of three independent experiments. **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403444&req=5

f5: Pontin is required for transcription of a subset of lincRNAs regulated by Oct4.(a) Proportions of the downregulated genes in Pontin-depleted ES cells out of those in Oct4-depleted ES cells. (b) Venn diagram of the downregulated lincRNAs in Oct4-depleted and Pontin-depleted ES cells. Sixteen lincRNAs are downregulated in both Pontin-depleted and Oct4-depleted ES cells. (c) List of 16 lincRNAs that are downregulated in both Oct4-depleted and Pontin-depleted ES cells. Asterisk (*) denotes the lincRNAs previously reported to function in repression of lineage specification process in ES cells. (d) Quantitative RT-PCR analysis of five lincRNAs involved in repression of differentiation processes among 16 lincRNAs that are downregulated in both Pontin-depleted and Oct4-depleted ES cells. The quantity of lincRNA was normalized by using primers to detect Gapdh. Values are expressed as mean±s.d. of three independent experiments. *P<0.05. (e) Luciferase assay was performed with reporters driven by linc1253 promoter possessing Oct4-binding elements (linc1253-1000) or deleted Oct4-binding region (linc1253-500). Luciferase activities were normalized by β-galactosidase activity. Values are expressed as mean±s.d. of three independent experiments. **p<0.01.
Mentions: Chromatin signature mapping and global gene expression analysis with RNAi screening in ES cells showed that lincRNAs play a role in the maintenance of self-renewal and pluripotency. As a subset of lincRNAs has been shown to be directly regulated by Oct4, we tested the possibility that Pontin regulates expression of Oct4-dependent lincRNAs. Our analysis of Oct4 targets affected by Pontin revealed that lincRNAs are significantly affected by Pontin expression (Fig. 5a). Among the known 226 lincRNAs expressed in ES cells, 54 lincRNAs are downregulated in Oct4-depleted ES cells and 16 (30% in Fig. 5a, P=2.80 × 10−3 by Fisher's exact test) of the 54 lincRNAs are also downregulated in Pontin-depleted ES cells (Fig. 5b and Supplementary Data 4). Interestingly, five lincRNAs (linc1253, linc1356, linc1517, linc1562 and linc1602) that are co-regulated by both Pontin and Oct4 from our analysis (denoted with asterisk in Fig. 5c) have been shown to function as repressors of lineage differentiation programme in ES cells18. Moreover, the genes that are upregulated by knockdown of linc1253, linc1356 or linc1517 significantly overlapped with those that are upregulated by Pontin depletion (false discovery rate<0.1) (Supplementary Fig. 4a). These analyses support that Pontin and lincRNAs may collaborate to repress lineage specification programmes in ES cells. Quantitative RT-PCR analysis confirmed that lack of either Oct4 or Pontin resulted in downregulation of transcript levels of five lincRNAs that function to repress lineage differentiation programme (Fig. 5d).

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