Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

The MLL gene was first identified because it is involved in chromosome translocations which produce novel fusion proteins that cause leukemia. The CXXC domain of MLL is a cysteine rich DNA binding domain with specificity for binding unmethylated CpG-containing DNA. The CXXC domain is retained in oncogenic MLL fusions, and is absolutely required for the fusions to cause leukemia. This project explored the role of the CXXC domain by introducing structure-informed point mutations within the MLL CXXC domain that disrupt DNA binding, and by performing domain swap experiments in which different CXXC domains from other proteins, including DNMT1, CGBP and MBD1, replaced the MLL CXXC domain in the context of an oncogenic MLL fusion. DNA binding experiments determined that the MLL CXXC domain has the highest DNA binding affinity, followed by the CGBP and DNMT1 CXXC domains. In vitro colony assays and in vivo murine leukemia assays were performed using isolated or transplanted murine bone marrow progenitor cells expressing mutant or wild type MLL-AF9 fusion proteins. MLL-AF9 containing the DNMT1 CXXC domain showed robust colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all showed reduced colony forming ability and leukemogenicity in vivo. The CXXC point mutations with reduced DNA binding also showed diminished in vitro colony formation and a loss of ability to cause leukemia in mice when present in MLL-AF9. Cell culture experiments with CXXC mutant fusion proteins showed that MLL-AF9 leukemogenicity correlates with the overexpression of MLL target genes and their protection from DNA methylation, but not with fusion protein localization to target genes. We conclude from the results of this study that the DNA binding activity of the MLL CXXC domain is essential to MLL fusion protein function. As the DNMT1 CXXC domain but not the CGBP CXXC domain could replace the MLL CXXC domain, this suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to the leukemogenic properties of MLL fusion proteins.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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