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

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.

Figure 1. Bptf mutants manifest early embryonic growth defects.

(A) Wild type (+/+), heterozygous (+/−), and homozygous (−/−)Bptf ^XG023 embryos at E5.5, E6.5, and E7.5 were removed from their decedua and genotyped using the PCR based method. Reduced growth rates are evident in the homozygous Bptf ^XG023 embryos as early as E5.5. (B) Hematoxylin and eosin (H&E) staining of E5.5, E6.5, and E7.5 mutant embryos show reduced growth of both embryonic and extra-embryonic tissues, the presence of visceral endoderm, and an absence of anterior–posterior asymmetry when compared to controls. Bptf mutant embryos were stained for phosphorylated histone H3, a marker of cell mitosis. Mutant embryos at E5.5, E6.5, and E7.5 have significantly reduced staining when compared to controls, suggesting that they have reduced cellular proliferation. Bptf-mutant embryos were stained for apoptotic cells using the TUNEL assay. Mutant embryos at E5.5, E6.5, and E7.5 do not have any positive staining by TUNEL when compared to controls, suggesting that they are not apoptotic. Scale Bars: E5.5 and E6.5 embryos = 50 µm, E7.5 embryos = 100 µm. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; m, mesoderm; xe, extra-embryonic ectoderm; xve, extra-embryonic visceral endoderm.

Figure 2. Bptf mutants are defective in the expression of distal visceral endoderm markers.

(A) Wild type embryos were stained in whole mount for Bptf mRNA by in situ RNA hybridization at E4.5, E5.5, and E6.5. Bptf is expressed in the inner cell mass and primitive endoderm at E4.5 and in the embryonic and extra-embryonic tissues in E5.5 and E6.5 embryos. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; xe, extra-embryonic ectoderm; DVE, distal visceral endoderm. (B) Whole mount in situ RNA hybridization analysis of wild type and mutant E4.5 and E5.5 embryos for Nanog, Gata6, Lefty1, Cer1, Hex1, and Nodal expression. At E4.5, Bptf mutant embryos show expression of Nanog, Gata6, Lefty1, and Hex1, suggesting that the primitive endoderm and inner cell mass is present in Bptf mutants. Mutant E5.5 embryos are defective in the expression of DVE markers Cer1 and Hex1, suggesting that mutants cannot form the DVE. Interestingly, the general visceral endoderm marker GATA6 is not expressed in the VE but rather in the embryonic ectoderm at E5.5.

Figure 3. Analysis of Bptf knockout mouse embryonic stem cells shows severe defects in gene expression during embryoid differentiation.

Relative expression of developmental markers during an embryoid body differentiation time course. The expression of many markers of the ectoderm (A), mesoderm (B), and endoderm (C) tissue lineages were severely defective in Bptf mutants during the differentiation time course.

Figure 4. Analysis of Bptf knockout mouse embryonic stem cells shows severe defects in gene expression during differentiation with RA and LIF withdrawal.

Bptf knockout embryonic stem (ES) cells were induced to differentiate using LIF withdrawal (LIF−) or the addition of retinoic acid (RA), and gene expression was monitored by microarray. (A) Bptf-dependent genes were defined as genes whose transcription increases or decreases more than 2-fold in the mutant compared to the wild type with a FDR value of <0.05. (B) A manual clustering analysis of Bptf-dependent genes by condition of dependence and expression values. Six expression categories were identified and include genes which are exclusively Bptf-dependent in LIF−, LIF+, or RA conditions, genes which are dependent in all conditions (constitutive regulation), genes which are dependent in 2 conditions (complex regulation), or genes which vary in direction of misregulation between conditions (mixed regulation). (C) Clustering analysis of Bptf-dependent genes important for the development of tissue lineages of the early embryo. Bptf is essential for the proper regulation of many markers of ectoderm, endoderm, and mesoderm tissue lineages.

Figure 5. 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.