III. Analysis of Cfp1 Functional Domains Required to Rescue Cytosine Methylation and in vitro Differentiation
1. DNA-binding activity of Cfp1 is not essential for appropriate global cytosine methylation
ES cells demonstrate a 60-80% decrease in global cytosine methylation in the absence of Cfp1 (Carlone 2005). The CXXC domain is a cysteine-rich domain that functions as the sole DNA-binding domain of Cfp1 (Lee 2001). Cfp1 binds specifically to unmethylated CpG motifs and localizes to euchromatin (Lee 2001; Lee 2002).
S. cerevisiae, S. pombe, and C. elegans lack cytosine methylation, and their respective Cfp1 homologues lack the CXXC domain. Therefore, the CXXC domain is expected to play a crucial role in the regulation of cytosine methylation. Consequently, experiments were performed to determine if DNA-binding activity of Cfp1 plays an essential role in the regulation of cytosine methylation. Global cytosine methylation was analyzed using a DNA methyl acceptance assay. This assay utilizes an in vitro methylation reaction to quantify transfer of radiolabled methyl groups from S-Adenosyl-L-Methionine onto genomic DNA sites that are not methylated (Fowler 1998). Therefore, the amount of radiolabeled methyl groups that are incorporated into a DNA sample is inversely related to the native DNA methylation status.
Consistent with previous data, DNA derived from CXXC1-/- or vector ES cells accepts approximately 3.5-fold more methyl groups than DNA derived from CXXC1+/+
ES cells, indicating a ~70% decrease in global cytosine methylation in cells lacking Cfp1 (Carlone 2005; Fig. 15). The reduced cytosine methylation observed in CXXC1-/-
FIGURE 15. Cfp1 has redundancy of function for rescue of global cytosine methylation.
Global cytosine methylation levels were determined by methyl acceptance assay for genomic DNA isolated from CXXC1+/+, CXXC1-/-, and CXXC1-/- ES cells expressing vector control, full-length Cfp1 (1-656), or the indicated Cfp1 truncations. The graph represents the results from three independent experiments with error bars representing standard error. Asterisks denote a statistically significant (p<0.05) difference compared to CXXC1-/- ES cells expressing full-length Cfp1 (1-656).
ES cells is rescued by expression of full-length Cfp1 protein (1-656) (Fig. 15).
Consistent with plating efficiency, expression of Cfp1 1-481 in CXXC1-/- ES cells (containing the PHD1, CXXC, acidic, basic, and coiled-coil domains), and Cfp1 302- 656 (containing the basic, coiled-coil, SID, and PHD2 domains) rescue global cytosine methylation (Fig. 15, Fig. 17). In addition, expression of either the amino half of Cfp1 (1-367; containing the PHD1, CXXC, acidic, and basic domains) or the carboxyl half of Cfp1 (361-597 and 361-656; containing the coiled-coil, SID, and PHD2 domains) is sufficient to rescue global cytosine methylation (Fig. 15, Fig. 16). Therefore, Cfp1 DNA-binding activity is not essential to maintain appropriate cytosine methylation levels, because Cfp1 361-597 and 361-656 rescue global cytosine methylation despite lacking the CXXC DNA-binding domain. In contrast, the basic domain is indispensable for Cfp1 1-367 rescue activity because removal of the basic domain (Cfp1 1-320) results in failure to rescue global cytosine methylation (Fig. 15).
In contrast to their inability to rescue plating efficiency, CXXC1-/- ES cells expressing Cfp1 103-481 (containing the CXXC, acidic, basic, and coiled-coil domains), or Cfp1 103-367 (containing the CXXC, acidic, and basic domains) rescue global cytosine methylation, indicating that the PHD1 domain is not essential for appropriate global cytosine methylation (Fig. 15, Fig. 17). In contrast, CXXC1-/- ES cells expressing Cfp1 318-481 (containing the basic, SID, and coiled-coil domains), Cfp1 318-412 (containing the basic and SID domains), or Cfp1 361-481 (containing the SID and coiled-coil domains) demonstrate decreased cytosine methylation, indicating that the PHD2 domain is required for cytosine methylation rescue activity of the C- terminal half of Cfp1 (Fig. 15, Fig. 17).
FIGURE 16. DNA-binding activity of Cfp1 or interaction with the Setd1 histone methyltransferase complexes is required for appropriate cytosine methylation.
Global cytosine methylation levels were determined by methyl acceptance assay for genomic DNA isolated from CXXC1+/+, CXXC1-/-, and CXXC1-/- ES cells expressing vector control, full-length Cfp1 (1-656), or the indicated Cfp1 mutations. The graphs represent three independent experiments with error bars representing standard error.
Asterisks denote a statistically significant (p<0.05) difference compared to full-length Cfp1 (1-656).
Consistent with plating efficiency, DNA isolated from CXXC1-/- ES cells expressing full-length Cfp1 that lacks DNA-binding activity (1-656 C169A or 1-656 C208A), or full-length Cfp1 that lacks interaction with the Setd1 histone H3K4 methyltransferase complexes (1-656 C375A or 1-656 YCS389AAA), rescue global cytosine methylation (Fig. 16, Fig. 17). However, ablation of DNA-binding activity of Cfp1 1-367 (1-367 C169A), or disruption of Cfp1 361-656 interaction with the Setd1 histone H3K4 methyltransferase complexes (361-656 C375A) results in failure to rescue global cytosine methylation (Fig. 16). In addition, failure to rescue global cytosine methylation was observed in DNA isolated from CXXC1-/- ES cells expressing full-length Cfp1 containing both point mutations (1-656 C169A, C375A or 1-656 C169A, YCS389AAA). Consistent with plating efficiency, this indicates that retention of either DNA-binding activity of Cfp1 or interaction of Cfp1 with the Setd1 H3K4 histone methyltransferase complexes is required for appropriate global cytosine methylation.