Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein - PubMed (original) (raw)

Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein

Diana L Carlone et al. Mol Cell Biol. 2005 Jun.

Abstract

Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP(-)(/)(-) cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP(-)(/)(-) embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP(-)(/)(-) cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP(-)(/)(-) cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP(-)(/)(-) phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.

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Figures

FIG. 1.

FIG. 1.

Generation of ES cell lines lacking CGBP. (A) Southern blot analysis was performed on genomic DNA isolated from ES cell clones derived from blastocysts resulting from heterozygous crosses. DNA was digested with NcoI and hybridized with a 500-bp KpnI/EcoRI probe downstream of the CGBP gene (10, 11). The arrows indicate the wild-type and disrupted alleles, and the deduced genotype at the CGBP locus is indicated above each lane. (B) Northern blot analysis was performed using total RNA isolated from either CGBP+/− or _CGBP_−/− ES cells and a 347-bp BamHI fragment probe derived from the murine CGBP cDNA (10). The arrow indicates the murine CGBP transcript of approximately 2.6 kb. As a control for loading, 28S and 18S rRNA bands were visualized by ethidium bromide staining. (C) Western blot analysis was performed on protein extracts isolated from ES cells carrying the indicated CGBP alleles using antiserum raised against CGBP (71). The blot was also incubated with an antiactin antibody as a loading control.

FIG. 2.

FIG. 2.

_CGBP_−/− ES cells exhibit an extended doubling time due to an elevated rate of apoptosis. (A) Growth rates were measured for ES cells carrying the indicated CGBP alleles. The curves for the CGBP+/+ and rescued _CGBP_−/cDNA cells are overlapping. (B) Exponentially growing ES cells of the indicated CGBP alleles were analyzed for cell cycle distribution by propidium iodide (PI) staining and flow cytometry. Numerical values represent the summary of data from three experiments. 2N and 4N represent diploid (G0-G1) and tetraploid (G2-M) genome content, respectively. (C) Exponentially growing ES cells of the indicated CGBP alleles were analyzed for apoptosis using Annexin V and propidium iodide staining and flow cytometry. UR, upper right panel corresponding to dead cells; LL, lower left panel corresponding to healthy cells; LR, lower right panel corresponding to apoptotic cells. Numerical values represent the summary of data from three experiments.

FIG. 3.

FIG. 3.

Failure of in vitro cellular differentiation in the absence of CGBP. (A) Colony morphology following induction of differentiation. Embryoid body outgrowths carrying the indicated CGBP alleles were cultured for 5 days in the absence of LIF. Magnification, ×20. (B) ES cells grown in the absence of LIF for 10 days were harvested, disaggregated, reseeded, and stained for alkaline phosphatase activity. One hundred cells of each genotype were scored for staining. Magnification, ×64. (C) ES cells carrying the indicated CGBP alleles were cultured for various times (0, 5, or 10 days) in the absence of LIF to induce differentiation. Total RNA was isolated, and reverse transcription-PCR was performed to examine the expression of lineage and development-specific gene markers. Oct4, marker of undifferentiated ES cells; Brachyury, mesoderm; MHC-β, muscle; c-fms, myeloid; gp-IIB, megakaryocyte; GATA-4, endoderm; HPRT, control for RNA quantity and integrity.

FIG. 4.

FIG. 4.

CGBP is essential for proper DNA methylation in murine ES cells. (A) Global cytosine methylation levels in ES cells carrying the indicated CGBP alleles were determined in the context of CCGG by thin-layer chromatography following digestion of genomic DNA with the restriction enzymes MspI or HpaII. A representative experiment is shown. (B) Summary of thin-layer chromatography data from three experiments. Error bars represent standard error, and asterisks denote a statistically significant (P < 0.05) difference compared to the wild type. Double asterisks denote a statistically significant (P < 0.05) difference between _CGBP_−/− cells and _CGBP_−/cDNA cells. (C) Global cytosine methylation levels in ES cells carrying the indicated CGBP alleles were determined by methyl acceptance assay. Error bars represent standard error, and asterisks denote a statistically significant (P < 0.05) difference compared to the wild type. Double asterisks denote a statistically significant (P < 0.05) difference between _CGBP_−/− cells and _CGBP_−/cDNA cells. The experiment was performed three times.

FIG. 5.

FIG. 5.

_CGBP_−/− ES cells exhibit reduced cytosine methylation at repetitive genomic elements. (A) Genomic DNA was isolated from ES cells carrying the indicated CGBP alleles, digested with MspI or HpaII, and Southern blot analysis was performed using a probe for the minor satellite repetitive element. The bracket indicates the region of low-molecular-weight bands that reflect cytosine hypomethylation. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading. (B) Same as in panel A, except the probe corresponds to the IAP retrovirus repetitive element. Arrows indicate bands that reflect hypomethylation. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading.

FIG. 6.

FIG. 6.

_CGBP_−/− ES cells exhibit reduced cytosine methylation at single-copy genes. (A) Genomic DNA was isolated from ES cells carrying the indicated CGBP alleles and digested with NcoI and HpaII, and Southern blot analysis was performed using a probe for the Rac2 gene (shown schematically). Arrows indicate a 2-kb band produced by HpaII and NcoI digestion of the fully methylated promoter and a 300-bp band produced by MspI digestion or by HpaII digestion if the restriction sites are unmethylated. The bent arrow indicates site of transcription initiation. N, NcoI; H, HpaII. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading. (B) Same as in panel A, except the genomic DNA was digested with HhaI and BamHI, and the probe corresponds to the _Pgk-_2 gene (shown schematically). The top three arrows indicate high-molecular-weight bands that correspond to heavy cytosine methylation which are dominant in CGBP+/+, CGBP+/−, and _CGBP_−/cDNA cells. The bottom four arrows indicate low-molecular-weight bands that reflect hypomethylation and increase in intensity in _CGBP_−/− and _CGBP_−/vector cells. The bent arrow indicates site of transcription initiation. B, BamHI. HhaI sites are denoted by hatch marks, and the question mark indicates a region of undetermined nucleotide sequence. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading.

FIG. 7.

FIG. 7.

_CGBP_−/− ES cells exhibit reduced cytosine methylation at imprinted genes. (A) Genomic DNA was isolated from ES cells carrying the indicated CGBP alleles and digested with HhaI and SacI, and Southern blot analysis was performed using a probe for the H19 gene (shown schematically). Arrows indicate position of paternally derived (imprinted) and maternally derived (nonimprinted) alleles. The bent arrow indicates site of transcription initiation. S, SacI; H, HhaI. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading. (B) Same as in panel A, except the DNA was digested with MluI and PvuII and the probe corresponds to the Igf2r locus (shown schematically). Arrows indicate position of maternally derived (imprinted) allele and paternally derived (nonimprinted) allele. P, PvuII. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading.

FIG. 8.

FIG. 8.

_CGBP_−/− ES cells exhibit reduced maintenance DNA methyltransferase activity. (A) Protein extracts were prepared from ES cells carrying the indicated CGBP alleles and assayed for in vitro DNA methyltransferase activity using poly(dI-dC), hemimethylated oligonucleotide, or unmethylated oligonucleotide substrates. Error bars represent standard error, and asterisks denote a statistically significant (P < 0.05) difference compared to the wild type. Double asterisks denote a statistically significant (P < 0.05) difference between _CGBP_−/− cells and _CGBP_−/cDNA cells. Each experimental value represents a summary from three experiments, with the exception of the analysis of CGBP+/+ and _CGBP_−/− extracts using the poly(dI-dC) substrate, which was performed seven times. (B) CGBP+/+ or _CGBP_−/− ES cells were transduced with the murine Moloney leukemia virus retrovirus. Genomic DNA was isolated at the indicated times following transduction and the provirus analyzed for cytosine methylation by Southern blot analysis. The structure of the provirus is illustrated schematically above the blot. H, HpaII; K, KpnI. Arrows indicate position of unmethylated and fully methylated provirus fragments.

FIG. 9.

FIG. 9.

_CGBP_−/− ES cells express reduced levels of Dnmt1 protein. Protein extracts isolated from ES cells carrying the indicated CGBP alleles were subjected to Western blot analysis using antiserum directed against Dnmt1, Dnmt3a, or Dnmt3b. An antiserum directed against actin was utilized as an internal control for protein loading. A representative experiment is presented, and the summary of data from numerous experiments is presented below. The number of replicates for each experimental value is indicated (N#). Error bars represent standard errors, and asterisks denote a statistically significant (P < 0.05) difference compared to the wild type. Double asterisks denote a statistically significant (P < 0.05) difference between _CGBP_−/− cells and _CGBP_−/cDNA cells.

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