V. Analysis of Cfp1 Function in DNA Damage Sensitivity
9. Cfp1 DNA-binding activity or interaction with the Setd1 complexes is required to rescue hypersensitivity to TMZ and cisplatin and Ape1 protein expression
Hypersensitivity to TMZ and cisplatin was observed in CXXC1-/- ES cells expressing Cfp1 mutations 1-367 C169A, 361-656 C375A, and 1-656 C169A, C375A (Fig. 54). In contrast, Cfp1 1-656 C169A and 1-656 C375A showed sensitivity to TMZ and cisplatin similar to that of CXXC1+/+ and CXXC1-/- ES cells expressing full-length Cfp1 1-656 (Fig. 54). These structure-function studies reveal that a point mutation
FIGURE 54. DNA-binding activity of Cfp1 or interaction with the Setd1 complexes is required to rescue DNA damage hypersensitivity.
Clonogenic survival for CXXC1+/+, CXXC1-/-, and CXXC1-/- ES cells expressing vector, full-length Cfp1 (1-656), and various Cfp1 mutations after treatment with temozolomide (A) or cisplatin (B). Each point represents the mean ± standard error from results of at least 3 experiments, with each experiment representing 3 dishes per treatment group. All data points located below asterisks denote statistically significant decreases compared to full-length Cfp1 (1-656).
FIGURE 55. Ape1 protein expression in CXXC1-/-ES cells expressing Cfp1 mutations.
Western blot analysis performed on protein extracts collected from CXXC1+/+, CXXC1-/-, vector control, and CXXC1-/- ES cells expressing full-length Cfp1 or the indicated Cfp1 mutations using antisera directed against Ape1 and β-actin as a loading control. The graph represents results from at least three independent experiments with
1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding activity of Cfp1 or association of Cfp1 with the Setd1 complexes is required to rescue hypersensitivity to TMZ and cisplatin.
10. Decreased Ape1 protein expression in Cfp1 mutations that exhibit hypersensitivity to DNA damaging agents
Western blot analysis was performed to determine the protein expression level of Ape1 in CXXC1-/- ES cells expressing Cfp1 mutations compared to CXXC1+/+ ES cells. Interestingly, Cfp1 mutations that exhibit hypersensitivity to TMZ and cisplatin (Cfp1 1-367 C169A, 361-656 C375A, and 1-656 C169A, C375A) also demonstrate a significant decrease in Ape1 protein expression (Fig. 55). In contrast, CXXC1-/- ES cells expressing Cfp1 mutations that rescue sensitivity to TMZ and cisplatin (Cfp1 1-656, 1-656 C169A, 1-656 C375A, 1-367, and 361-656) also rescue Ape1 protein expression (Fig. 55). This reveals a correlation between Ape1 protein expression levels and sensitivity of CXXC1-/- ES cells expressing Cfp1 mutations to DNA damaging agents.
11. Summary
The results presented in this portion of the dissertation demonstrate the
importance of Cfp1 for DNA damaging agent sensitivity and Ape1 protein expression.
In the absence of Cfp1, ES cells demonstrate hypersensitivity to a variety of DNA damaging agents, including IR, etoposide, MMS, TMZ, cisplatin, and hydrogen peroxide but do not demonstrate hypersensitivity to non-genotoxic agents MTX and taxol. DNA damage hypersensitivity is rescued upon expression of Cfp1 in CXXC1-/- ES cells. In contrast, DNA damage hypersensitivity was not observed in DNMT1-/- ES cells. CXXC1-/- ES cells exhibit ~50% decrease in protein expression of Ape1 and ~45-50% decrease in Ape1 endonuclease activity, a dual function enzyme involved in DNA base excision repair and redox activation of transcription factors. In contrast, DNMT1-/- ES cells exhibit a slight increase in Ape1 protein expression and endonuclease activity. In addition, CXXC1-/- ES cell DNA incorporates more platinum, and CXXC1-/- ES cells accumulate increased amounts of H2AX-γ after treatment with TMZ and cisplatin. Structure-function analysis revealed that either the amino half of Cfp1 (amino acids 1-367) or carboxyl half of Cfp1 (amino acids 361-656) are sufficient to rescue the hypersensitivity to DNA damaging agents and decreased Ape1 protein expression, demonstrating that Cfp1 contains redundant functional domains. Additional structure-function studies revealed that retention of either DNA-binding activity or interaction with the Setd1A and Setd1B histone H3K4 methyltransferase complexes is required to rescue the hypersensitivity to DNA damaging agents and decreased Ape1 protein expression. A summary of the DNA damage agent sensitivity and Ape1 protein expression rescue activity of CXXC1-/- ES cells expressing Cfp1 mutations is presented in Table 10.
- -
55 1084 18B
- -
84 1084Cys18A
361-656 C375A
+ +
58 905-6
+ +
57 905-2
361-656
- -
129 1-367 2A
- -
89 1-367 1C
1-367 C169A
+ +
63 1101-8
+ +
91 1101-4
1-367
- -
54 1-656 CC2A
- -
94 1-656 CC3A
1-656 C169A,C375A
+ +
60 Cys1 5
+ +
102 Cys1 1D
1-656 C375A
+ +
104 1-656 7A
+ +
40 PFCX6
1-656 C169A
+ +
111 PFCGBP2
+ +
108 PFCGPB1
1-656
- -
NA Evec1
- -
NA EVec2
Vector
APE1 protein TMZ, Cisplatin
sensitivity CFP1 protein
%CXXC1+/+
clone
- -
55 1084 18B
- -
84 1084Cys18A
361-656 C375A
+ +
58 905-6
+ +
57 905-2
361-656
- -
129 1-367 2A
- -
89 1-367 1C
1-367 C169A
+ +
63 1101-8
+ +
91 1101-4
1-367
- -
54 1-656 CC2A
- -
94 1-656 CC3A
1-656 C169A,C375A
+ +
60 Cys1 5
+ +
102 Cys1 1D
1-656 C375A
+ +
104 1-656 7A
+ +
40 PFCX6
1-656 C169A
+ +
111 PFCGBP2
+ +
108 PFCGPB1
1-656
- -
NA Evec1
- -
NA EVec2
Vector
APE1 protein TMZ, Cisplatin
sensitivity CFP1 protein
%CXXC1+/+
clone
TABLE 10. Summary of TMZ and cisplatin sensitivity and Ape1 protein expression rescue.
The table presents a summary of the rescue data for CXXC1-/- ES cells
expressing indicated Cfp1 mutations. Ability to rescue the indicated defect is indicated with (+), inability to rescue (significant [p<0.05] difference compared to Cfp1 1-656) is indicated with (-), and NA represents not applicable.