Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biochemistry Program

Abstract

Rearrangements of the MLL gene at chromosome band 11q23 have been associated with a heterogeneous group of lymphoid, myeloid and mixed lineage leukemias. MLL rearrangements occur approximately in 70% of infant leukemias and are also common in therapy-related leukemias where patients were previously treated with topoisomerase II inhibitors. Unfortunately, these patients have a poor prognosis. MLL gene rearrangements give rise to chimeric proteins that contain the N-terminal portion of MLL fused to the C-terminal portion of over 50 different fusion partners. The chimeric proteins cause constitutive expression of some MLL target genes such as HOXA9 and MEIS1, and enhanced proliferation of hematopoietic progenitors. MLL chimeras do not retain the histone methyltransferase activity of wild type MLL and it is unclear how they deregulate the expression of MLL target genes.

AF9 is one of the most common MLL fusion partners encountered in patients with leukemias. Moreover, Af9 is known to negatively regulate the expression of the epithelial sodium channel α (ENaCα) gene in murine renal collecting ducts by modulating the activity of the Dot1l. It has been shown that Af9-Dot1l promotes H3K79 methylation at specific sites in the ENaCα promoter, which then contributes to its repressed state. A number of reports have described the direct or indirect association of

the C-terminus of AF9 with several distinct proteins including activators as well as repressors of transcription apart from DOT1L. Our data indicate that other AF9 binding proteins compete with DOT1L thereby diminishing its activity. Specifically, we show that AF9 is part of a protein multimer containing members of Polycomb group PRC1 complex, CBX8, RING1B, and BMI1. The interaction of AF9 with other PRC1 proteins is directly mediated by CBX8 and precludes AF9-DOT1L binding. Knockdown of CBX8 with shRNA leads to decreased ENaCα mRNA levels. In contrast, CBX8 overexpression results in increased ENaCα mRNA levels and this effect can be partially overcome by co-overexpression of AF9. As predicted, expression of CBX8 is accompanied by decreased H3K79 trimethylation at ENaCα promoter regions. Thus, our data suggest that changing the abundance of different AF9 binding partners shifts the equilibrium of functionally distinct AF9 complexes and alters the regulation of target genes. Moreover, although CBX8 is part of the repressive PRC1 Polycomb complex, its increased expression may actually increase the expression of genes normally repressed by DOT1L through its interaction with AF9.

Furthermore, it has been shown that AF4 and AF9 exist in a large multiprotein complex containing positive transcription elongation factor b (P-TEFb) and therefore are linked to transcriptional elongation. To understand the significance of the AF9-AF4 interaction, our laboratory has mapped the domains involved in their binding and has developed synthetic peptides (PFWT and SPK-111) capable of disrupting their interaction in vitro and in vivo. The peptides mimic the amino acid sequence of the AF9 binding domain within AF4. These peptides are toxic to MLL-AF4 and MLL-AF9 fusion harboring leukemic cell lines, suggesting the importance of the AF9-AF4 interaction for the survival of these leukemic cells. However, the molecular mechanism of action of peptide is still not known.

We showed that after peptide treatment, MV4-11 (MLL-AF4) and MOLM13 (MLL-AF9) leukemic cells have reduced levels of MEIS1 and MYC expression both at the transcript levels and protein levels. MEIS1 and c-MYC are known targets of MLL fusion proteins. Moreover, the reduced MYC transcript levels correlate with the decreased recruitment of cyclin dependent kinase 9 (CDK9), a component of P-TEFb and phosphorylation of Ser2 of C-terminal domain (CTD) of RNA PolII. Hence, our data suggest that peptide mediated disruption of AF9-AF4 interaction interferes with the stable complex formation involving P-TEFb, which further impairs the productive elongation of the transcripts.

Therefore, modulation of protein-protein interactions involving AF9 would be predicted to have important effects on disease processes that subvert AF9, including MLL-AF9 leukemias.

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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