HOX11 is a homeodomain transcription factor that has been implicated in leukaemic transformation associated with T-cell acute lymphoblastic leukaemia (T-ALL). Its role in leukaemogenesis remains enigmatic, nevertheless, in vitro and in vivo studies have provided additional evidence supporting the role of HOX11 as an oncogene. The mechanism by which HOX11 transforms cells is yet to be elucidated, however, HOX11 has been postulated to function by binding regulatory elements within the promoter regions of specific target genes in order to control gene transcription. The identification of transcriptional targets is thus thought to be critical to our understanding of the pathways controlled by this master gene regulator. To date, only three candidate HOX11 target genes have been reported and given that HOX11 overexpression can have a profound impact on cell behaviour, it is likely that many more exist. In this study, we sought to further understand the role of HOX11 in tumorigenesis by: 1) The identification of novel putative HOX11 target genes by profiling gene expression in response to HOX11 in a number of cell lines using a combination of RDA, cDNA microarray and GeneChip approaches and 2) confirming target gene status by assessing whether the proximal promoters of the leading candidates identified are transcriptionally regulated by HOX11. To identify genes whose expression was altered by HOX11, three techniques were employed, namely representational difference analysis, cDNA microarray and Affymetrix GeneChip array. Because of the relative novelty of these technologies, all three methods were employed in a complementary manner. While representational difference analysis did not require dedicated equipment and enabled the identification of novel genes, the technique was labour-intensive and also exhibited a number of problems including high levels of background. Emphasis was therefore placed on the more systematic microarray approaches that enabled a global investigation of expression patterns and thus the identification of a range of candidate target genes. Initially, this involved cDNA microarray experiments, however, during the course of this work Affymetrix GeneChip technology became available. The latter was identified as the most appropriate technology for the identification of candidate target genes because of its relative ease of use, as well as its employment of multiple independent probe pairs which greatly improved background noise, increased the range and accuracy of detection, minimized the effects of cross hybridization and drastically reduced the rate of false positives and miscalls. Using these combined approaches, several genes of interest were identified which were differentially regulated in the presence of HOX11 and thus may represent oncogenically or physiologically relevant target genes. These included OSTEOPONTIN, PAG, GUANOSINE DIPHOSPHATE DISSOCIATION INHIBITOR 3, SUR8, GAS3, C-KIT, VEGFC, NOR1 and SMARCD3. In order to confirm their role as target genes, four candidates (C-KIT, VEGFC, NOR1 and SMARCD3) were characterized in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of C-KIT and VEGFC by HOX11 was observed, which provided further evidence for their status as target genes. This repression was, however, in stark contrast to the transcriptional activation seen when the C-KIT and VEGFC proximal promoters were co-transfected with a HOX11 mutant lacking the third helix of the DNA-binding homeodomain. This unexpected finding suggested that the transcriptional activity of HOX11 is complex and highly context-dependent, and in particular, highlighted the importance of an intact homeodomain for HOX11 function. C-KIT and VEGFC are both involved in tyrosine kinase signal transduction pathways, as a receptor tyrosine kinase and tyrosine kinase ligand, respectively. C-KIT plays an important role in the survival and self-renewal of haematopoietic cells. It is a previously identified and relatively well characterized oncogene known to be regulated by other transcription factors (SCL/TAL1 and LMO) implicated in the pathogenesis of T-ALL. VEGFC is a member of the vascular endothelial growth factor family that functions in angiogenesis and lymphangiogenesis. A paracrine loop involving VEGFC and its receptor VEGFR-3 has previously been implicated in leukaemic cell survival. While further work is required in order to confirm the status of VEGFC and C-KIT as oncogenically-relevant HOX11 target genes and to characterize their exact mode of regulation, these findings implicate receptor tyrosine kinases in HOX11-mediated tumorigenesis and underscore their potential importance as therapeutic targets in haematological malignancies.

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.

HOX11 is a developmental regulator that plays a crucial role in the normal development of the spleen and is also aberrantly activated by the t(10;14)(q24;q11) and variant t(7;10)(q35;q24) translocations in a subset of T-cell acute lymphoblastic leukaemias (TALLs). The recent finding that HOX11 is deregulated in up to 40% of childhood TALLs when abnormalities not detected by cytogenetics are included, suggests that the over-expression of HOX11 and subsequent deregulation of downstream target genes are critical events in the progression of this tumour type. To date, three candidate HOX11 target genes have been reported, two of which are Aldehyde Dehydrogenase 1a1 (ALDH1A1) and Four and a Half LIM domain Protein 1 (FHL1). This investigation focused on two aspects of HOX11 function, namely its roles as a transcriptional regulator and as a nuclear oncoprotein capable of inducing neoplastic transformation. More specifically, we sought to further understand the role of HOX11 in tumorigenesis by 1) Confirming target gene status of ALDH1A1 and FHL1 by assessing whether their proximal promoter regions are transcriptionally regulated by HOX11, 2) Investigating the regulatory elements/transcriptional complexes involved in the response of ALDH1A1 to HOX11 in both a T-cell and an erythroid cell line in order to gain an insight into the mechanism(s) responsible for mediating a HOX11 activity and 3) Assessing the ability of ALDH1A1 and FHL1 to perturb normal patterns of haematopoiesis, on the basis that the transforming capabilities of HOX11 are thought to derive from its ability to affect haematopoietic cell differentiation. To confirm ALDH1A1 and FHL1 as target genes, they were both characterised in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of ALDH1A1 and FHL1 by HOX11 was observed in PER-117 T-cells which provided further evidence for their status as target genes. In the case of ALDH1A1, a CCAAT box (-74/-70bp) was identified as the primary cis-regulatory element involved in ALDH1A1 transcription and repression by HOX11 appeared to occur, either directly or indirectly, via interactions at the CCAAT box. Electromobility shift assays (EMSAs) revealed the disruption of a specific complex at this site by HOX11, which also altered the formation of complexes at a non-canonical TATA box (a GATA box at -34/-29bp). Significantly, HOX11 was shown to have the potential to interact with TFIIB, a member of the basal transcriptional complex. This, together with the presence of a TFIIB responsive element immediately 5’ of the GATA box, suggested that HOX11 may repress transcription by interfering with members of a preinitiation complex on the ALDH1A1 promoter. The transcriptional repression by HOX11 demonstrated in T-cells was dependent on DNA binding helix 3 of the homeodomain, suggesting that repression may require DNA binding. Alternatively, this region may be required for stable protein-protein interactions. In support of this, the in vitro association of HOX11 with TFIIB was disrupted upon deletion of helix 3, and the HOX11∆H3 mutant switched from a transcriptional repressor to a potent activator of transcription. Together, this data supports a model whereby HOX11 represses transcription by interfering with activation complexes at the CCAAT box and at the GATA box possibly via protein-protein interactions involving the homeodomain helix 3, whereas deletion of the region disables repressor-specific interactions, resulting in potent activation by HOX11. Luciferase reporter gene assays investigating the response of nested deletions of the ALDH1A1 promoter to HOX11 in the HEL900 erythroleukaemic cell line, also identified the CCAAT box (-74/-70bp) as the primary cis-regulatory element involved in ALDH1A1 transcription. However, in stark contrast to the its effect in T-cells, HOX11 was shown to activate transcription in the HEL cell line, both from the empty pGL3Basic luciferase reporter vector and from the ALDH1A1 promoter, in a manner independent of the homeodomain DNA binding helix 3. HOX11 thus appears to be a dichotomous regulator, capable of both transcriptional activation and repression depending on the circumstances. The mechanisms underlying these two functions are also appear to be distinct, with repression but not activation requiring the presence of homeodomain helix 3. ALDH1A1 encodes an enzyme involved in the irreversible conversion of retinaldehyde to the biologically active metabolite, retinoic acid (RA) and appears to be physiologically regulated by Hox11 in the developing spleen. Since RA is a potent modulator of cellular differentiation, proliferation and apoptosis, the dysregulation of RA synthesis is likely to have severe consequences for the cell and may constitute a mechanism whereby overexpression of HOX11 predisposes T-cells to malignant transformation. FHL1 also appears to have potential relevance to tumorigenesis, given that it encodes protein isoforms with suspected roles in transcriptional regulation. As a starting point to investigate a possible link between these HOX11 target genes and leukaemogenesis, the effect of overexpressing ALDH1A1 and FHL1 on murine haematopoiesis was assessed following reconstitution of lethally irradiated mice with retrovirally-transduced primary murine bone marrow cells. The enforced expression of ALDH1A1 in bone marrow was associated with a marked increase in myelopoiesis and a decrease in B and T-lymphopoiesis. By contrast, overexpression of FHL1 was not associated with perturbations in myelopoiesis or lymphopoiesis, although a slight increase in erythropoiesis was observed in the bone marrow. While further work is required to clarify the possible oncogenic roles of both of these HOX11 target genes, these findings have served to identify ALDH1A1 in particular, as a gene which could potentially be involved in HOX11-mediated tumorigenesis.

Haematological cancers like leukemia and lymphoma are characterized by genetic abnormalities, specifically chromosomal translocations. Analyses of the translocation breakpoint regions in patients have shown that some loci in the genome are more susceptible to breakage than others. However, very little is known about the mechanism of generation of many such chromosomal translocations. In the present study, we have attempted to understand the mechanism of fragility of three regions, which are prone to breaks during translocations in follicular lymphoma (FL) and T-cell leukemia. The t(14;18) translocation in FL is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3’ UTR of the BCL2 gene and are broadly classified into three clusters, namely major breakpoint region (mbr), minor breakpoint cluster region (mcr) and the intermediate cluster region (icr). The RAG complex has been shown to cleave BCL2 mbr by recognizing an altered DNA structure. In the present study, by using a gel based assay, nature of the non-B DNA structure at BCL2 mbr was identified as parallel intramolecular G-quadruplex. Various studies including circular dichroism (CD), mutagenesis, DMS modification assay and 1H NMR showed the presence of three guanine tetrads in the structure. Further, evidence was also found for the formation of such a G-quadruplex structure within mammalian cells. In an effort to characterize the mechanism of fragility of mcr, a unique pattern of RAG cleavage was observed in a sequence dependent manner. Three independent nicks of equal efficiency were generated by RAGs at the cryptic sequence, “CCACCTCT”, at mcr and at a cytosine upstream of it, unlike a single specific nick at the 5’ of heptamer during V(D)J rearrangement. Interestingly, RAG nicking at mcr occured in the presence of both Mg2+ and Mn2+. Using recombination assay, followed by sequencing of the junctions, we find that mcr can recombine with standard RSS in vivo, albeit at a very low frequency. Mutations to this novel motif abolish recombination at the mcr within the cells. In order to determine the prevalence of t(14;18) translocation in the healthy Indian population, nested PCR approach followed by Southern hybridization was used. Results showed 34% prevalence of t(14;18) translocation in the Indian population. Although, no gender based difference was observed, an age dependent increase was found in adults. Further, presence of the t(14;18) transcripts was also detected. The mechanism underlying the fragility of the t(10;14) translocation involving HOX11 gene in T-cell leukemia is not known. Using primer extension assays on a plasmid DNA containing HOX11 breakpoint region, presence of consistent pause sites corresponding to two G-quadruplex forming regions, flanking the patient breakpoints, were detected. These replication blocks were dependent on K+ ions. Native gel shift assays, mutation analysis, S1 nuclease and CD, further revealed formation of intermolecular G-quadruplexes, unlike the BCL2 mbr. Further, sodium bisulfite modification assay indicated the presence of such structures in the genomic DNA within cells. Hence, we propose that two independent G-quadruplex structures formed in the HOX11 gene could interact with each other, thereby resulting in fragility of the intervening sequences, where majority of the patient breakpoints are mapped. Overall, this study has attempted to understand the role of both sequence and structure of DNA, in generating chromosomal fragility during t(14;18) translocation in FL and t(10;14) translocation in T-cell leukemia. These results may facilitate future studies in unraveling the mechanism leading to genomic instability in other lymphoid cancers.