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Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

Landry J, Sharov AA, Piao Y, Sharova LV, Xiao H, Southon E, Matta J, Tessarollo L, Zhang YE, Ko MS, Kuehn MR, Yamaguchi TP, Wu C - PLoS Genet. (2008)

Bottom Line: Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent.We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors.We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Cell Biology, National Cancer Institute, National Institutes of Heath, Bethesda, MD, USA. landrjos@mail.nih.gov

ABSTRACT
We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

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Bptf is necessary for Smad mediated gene regulation.(A) RT-PCR analysis of known Smad target genes from activin-A induced wild type (+/+) and Bptf knockout (−/−) embryonic stem (ES) cells shows Cer1, T, Gsc to be dependent, and Fgf8, Lefty1 to be partially dependent on Bptf for full activation. (B) Like CBP/p300, Bptf regulates Smad-dependent genes in vivo. Bptf or CBP/p300 were knocked down in activin-A induced or uninduced ES cells. The expression of gene targets was determined by RT-PCR and is expressed as a ratio of induced/uninduced for each knockdown condition. Lefty1, Fgf8, T, Gsc, and Cer1 require both Bptf and CBP/p300 for full activin-A–dependent gene activation. (C) NURF interacts with the Smad transcription factors in vitro. Recombinant NURF complex was subjected to GST pull-down assays using resin bound GST, GST-Smads, GST-Smad fragments, and GST-β-catenin. GST-Smad2 and 3 but not GST and GST-β-catenin controls can specifically pull down the NURF complex. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the NURF complex. The C-terminal domain of Smad2 interacts with native NURF from crude nuclei extracts. High salt nuclei extracts from ES cells was subjected to GST pull-down assays using resin bound GST and GST-Smad fragments. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the Bptf and Snf2h/l components of the native NURF complex and the histone acetyl-transferase CBP from nuclei extracts. (D) Chromatin immunoprecipation of Snf2h/l and histone modifications 3me-K4H3 and 3me-K27H3 at Lefty1 show recruitment of the Snf2h/l subunit to the neural plate specific enhancer (NPE), a region which contains Smad binding elements, in a Bptf and an activin-A–dependent manner. Snf2h/l ChIP is expressed as a ratio of induced (+activin-A) to uninduced (−activin A). Histone modifications are shown during induced (+activin-A) conditions for +/+ and −/− cells and have been normalized to a pan histone H3 pulldown. NPE = neural plate specific enhancer, LPE = lateral plate specific enhancer, RSS = right side specific enhancer.
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pgen-1000241-g005: Bptf is necessary for Smad mediated gene regulation.(A) RT-PCR analysis of known Smad target genes from activin-A induced wild type (+/+) and Bptf knockout (−/−) embryonic stem (ES) cells shows Cer1, T, Gsc to be dependent, and Fgf8, Lefty1 to be partially dependent on Bptf for full activation. (B) Like CBP/p300, Bptf regulates Smad-dependent genes in vivo. Bptf or CBP/p300 were knocked down in activin-A induced or uninduced ES cells. The expression of gene targets was determined by RT-PCR and is expressed as a ratio of induced/uninduced for each knockdown condition. Lefty1, Fgf8, T, Gsc, and Cer1 require both Bptf and CBP/p300 for full activin-A–dependent gene activation. (C) NURF interacts with the Smad transcription factors in vitro. Recombinant NURF complex was subjected to GST pull-down assays using resin bound GST, GST-Smads, GST-Smad fragments, and GST-β-catenin. GST-Smad2 and 3 but not GST and GST-β-catenin controls can specifically pull down the NURF complex. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the NURF complex. The C-terminal domain of Smad2 interacts with native NURF from crude nuclei extracts. High salt nuclei extracts from ES cells was subjected to GST pull-down assays using resin bound GST and GST-Smad fragments. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the Bptf and Snf2h/l components of the native NURF complex and the histone acetyl-transferase CBP from nuclei extracts. (D) Chromatin immunoprecipation of Snf2h/l and histone modifications 3me-K4H3 and 3me-K27H3 at Lefty1 show recruitment of the Snf2h/l subunit to the neural plate specific enhancer (NPE), a region which contains Smad binding elements, in a Bptf and an activin-A–dependent manner. Snf2h/l ChIP is expressed as a ratio of induced (+activin-A) to uninduced (−activin A). Histone modifications are shown during induced (+activin-A) conditions for +/+ and −/− cells and have been normalized to a pan histone H3 pulldown. NPE = neural plate specific enhancer, LPE = lateral plate specific enhancer, RSS = right side specific enhancer.

Mentions: Accordingly, we monitored the dependence of Smad-responsive genes on the presence of Bptf in ES cells. ES cells readily responded to activin-A as monitored by the phosphorylation of Smad2 (Figure S22A, Figure S22B). From these experiments we identified a number of Smad-dependent genes which completely or partially require Bptf for full activation. Genes requiring Bptf for full activation include Cer1, Gsc and T (Figure 5A). Genes which partially require Bptf include FGF8, Lefty1 and p21 (Figure 5A) (Figure S23).


Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

Landry J, Sharov AA, Piao Y, Sharova LV, Xiao H, Southon E, Matta J, Tessarollo L, Zhang YE, Ko MS, Kuehn MR, Yamaguchi TP, Wu C - PLoS Genet. (2008)

Bptf is necessary for Smad mediated gene regulation.(A) RT-PCR analysis of known Smad target genes from activin-A induced wild type (+/+) and Bptf knockout (−/−) embryonic stem (ES) cells shows Cer1, T, Gsc to be dependent, and Fgf8, Lefty1 to be partially dependent on Bptf for full activation. (B) Like CBP/p300, Bptf regulates Smad-dependent genes in vivo. Bptf or CBP/p300 were knocked down in activin-A induced or uninduced ES cells. The expression of gene targets was determined by RT-PCR and is expressed as a ratio of induced/uninduced for each knockdown condition. Lefty1, Fgf8, T, Gsc, and Cer1 require both Bptf and CBP/p300 for full activin-A–dependent gene activation. (C) NURF interacts with the Smad transcription factors in vitro. Recombinant NURF complex was subjected to GST pull-down assays using resin bound GST, GST-Smads, GST-Smad fragments, and GST-β-catenin. GST-Smad2 and 3 but not GST and GST-β-catenin controls can specifically pull down the NURF complex. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the NURF complex. The C-terminal domain of Smad2 interacts with native NURF from crude nuclei extracts. High salt nuclei extracts from ES cells was subjected to GST pull-down assays using resin bound GST and GST-Smad fragments. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the Bptf and Snf2h/l components of the native NURF complex and the histone acetyl-transferase CBP from nuclei extracts. (D) Chromatin immunoprecipation of Snf2h/l and histone modifications 3me-K4H3 and 3me-K27H3 at Lefty1 show recruitment of the Snf2h/l subunit to the neural plate specific enhancer (NPE), a region which contains Smad binding elements, in a Bptf and an activin-A–dependent manner. Snf2h/l ChIP is expressed as a ratio of induced (+activin-A) to uninduced (−activin A). Histone modifications are shown during induced (+activin-A) conditions for +/+ and −/− cells and have been normalized to a pan histone H3 pulldown. NPE = neural plate specific enhancer, LPE = lateral plate specific enhancer, RSS = right side specific enhancer.
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pgen-1000241-g005: Bptf is necessary for Smad mediated gene regulation.(A) RT-PCR analysis of known Smad target genes from activin-A induced wild type (+/+) and Bptf knockout (−/−) embryonic stem (ES) cells shows Cer1, T, Gsc to be dependent, and Fgf8, Lefty1 to be partially dependent on Bptf for full activation. (B) Like CBP/p300, Bptf regulates Smad-dependent genes in vivo. Bptf or CBP/p300 were knocked down in activin-A induced or uninduced ES cells. The expression of gene targets was determined by RT-PCR and is expressed as a ratio of induced/uninduced for each knockdown condition. Lefty1, Fgf8, T, Gsc, and Cer1 require both Bptf and CBP/p300 for full activin-A–dependent gene activation. (C) NURF interacts with the Smad transcription factors in vitro. Recombinant NURF complex was subjected to GST pull-down assays using resin bound GST, GST-Smads, GST-Smad fragments, and GST-β-catenin. GST-Smad2 and 3 but not GST and GST-β-catenin controls can specifically pull down the NURF complex. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the NURF complex. The C-terminal domain of Smad2 interacts with native NURF from crude nuclei extracts. High salt nuclei extracts from ES cells was subjected to GST pull-down assays using resin bound GST and GST-Smad fragments. The GST-Smad2C containing the C-terminal fragment of Smad2 specifically pulls down the Bptf and Snf2h/l components of the native NURF complex and the histone acetyl-transferase CBP from nuclei extracts. (D) Chromatin immunoprecipation of Snf2h/l and histone modifications 3me-K4H3 and 3me-K27H3 at Lefty1 show recruitment of the Snf2h/l subunit to the neural plate specific enhancer (NPE), a region which contains Smad binding elements, in a Bptf and an activin-A–dependent manner. Snf2h/l ChIP is expressed as a ratio of induced (+activin-A) to uninduced (−activin A). Histone modifications are shown during induced (+activin-A) conditions for +/+ and −/− cells and have been normalized to a pan histone H3 pulldown. NPE = neural plate specific enhancer, LPE = lateral plate specific enhancer, RSS = right side specific enhancer.
Mentions: Accordingly, we monitored the dependence of Smad-responsive genes on the presence of Bptf in ES cells. ES cells readily responded to activin-A as monitored by the phosphorylation of Smad2 (Figure S22A, Figure S22B). From these experiments we identified a number of Smad-dependent genes which completely or partially require Bptf for full activation. Genes requiring Bptf for full activation include Cer1, Gsc and T (Figure 5A). Genes which partially require Bptf include FGF8, Lefty1 and p21 (Figure 5A) (Figure S23).

Bottom Line: Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent.We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors.We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Cell Biology, National Cancer Institute, National Institutes of Heath, Bethesda, MD, USA. landrjos@mail.nih.gov

ABSTRACT
We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

Show MeSH
Related in: MedlinePlus