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Investigation of the molecular function of the nuclear oncoprotein HOX11 in human t-cell leukaemia

Heidari, Mansour (2003) Investigation of the molecular function of the nuclear oncoprotein HOX11 in human t-cell leukaemia. PhD thesis, Murdoch University.

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HOXll, the prototypical member of the HOXll family (HOX11, HOXllLl and HOXllL2) was originally discovered as a transcriptional regulator aberrantly expressed in tumours with an immature T-cell phenotype (T-ALL) as a result of specific chromosomal translocations involving T-cell receptor loci. Subsequently, it was revealed that HOXll is required for normal spleen development since newborn Hoxll-/- mice exhibit asplenia. In both its normal and abnormal roles, HOXll has been postulated to function by binding regulatory elements within specific target genes to control gene transcription. However, very few genomic targets of HOX11 have been identified and little is known about its mode of action. In this study, we sought to further understand the role of HOX11 in controlling differentiation and cell growth by 1) determining the identity of genomic sequences that are directly bound by HOXll and 2) determining the identity of proteins which exist within HOXll-containing nuclear complexes.

To identify direct HOXll target sequences, a whole genome PCR-based screening method was employed using immobilised recombinant HOXll that had first been expressed as a biologically active GST fusion protein. Using this approach, restriction enzyme-cleaved human genomic DNA was selected for high-affinity HOXll binding sites. Unexpectedly, almost all clones isolated contained sequences derived from satellite 2 DNA that, together with related satellite 3 DNA, is found on most chromosomes at transcriptionally inactive pericentromeric heterochromatin. The specific binding of HOXl1 to satellite 2 DNA was verified by bandshift assays using both recombinant HOXll protein and nuclear extract derived from the T-ALL cell line, ALL-SIL. DNA-protein complexes containing HOX11 were identified by their ablation upon addition of HOXl1 antibody.

To confirm that HOXll associates with pericentromeric heterochromatin in vivo, HOXll was characterised in terms of its nuclear localisation during interphase in unsynchronised leukaemic T-cells (ALL-SIL) harbouring a translocation involving the HOXll locus. Using indirect immunofluorescence and confocal microscopy, HOXll antibody produced a punctate pattern of staining in the nucleus with discrete areas of dense staining superimposed on a diffuse distribution of HOXll protein. By dual staining, the bright HOXll foci correlated with centromeres since they overlapped with signals detected by an antibody specific for the centromeric protein CENP-B. Further evidence for a direct interaction of HOXll with satellite 2 DNA was provided by chromatin immunoprecipitation assay. In the presence of HOXll antibody, DNA fragments containing satellite 2 sequences were irnmunoprecipitated from sheared, cross-linked ALL-SIL chromatin but not from chromatin isolated from the HOXll-negative T-cell line PER-1 17. Finally, using a combination of immunoprecipitation with HOXll antibody, gel electrophoresis and mass peptide fingerprinting, a set of nuclear proteins were identified as potential HOXll interactors which are known to either localise to centromeric regions or act as regulators of gene expression. Together, these results implicate HOXl 1 in a functional interaction with centromeric heterochromatin, which may be a key feature of this oncoprotein in terms of both its T-cell transformation and transcriptional regulatory functions.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Greene, Wayne
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