Molecular cloning of the t(10;14)(q24;q11) recurrent breakpoint of T cell acute lymphoblastic leukemia has demonstrated a transcript for the candidate gene TCL3. Characterization of this gene from chromosome segment 10q24 revealed it to be a new homeobox, HOX11. The HOX11 homeodomain is most similar to that of the murine gene Hlx and possesses a markedly glycine-rich variable region and an acidic carboxyl terminus. HOX11, while expressed in liver, was not detected in normal thymus or T cells. This lineage-restricted homeobox gene is deregulated upon translocation into the T cell receptor locus where it may act as an oncogene (Hatano, 1991).
The translocation t(10;14)(q24;q11) is an acquired change seen in 4% to 7% of T-cell acute lymphoblastic leukemias (T-ALL). The translocation juxtaposes the T-cell receptor (TCR) delta-chain gene in chromosome 14q11 with a novel region in chromosome 10q24 and is likely catalyzed by recombinases normally involved in the generation of immunoglobulin and TCR diversity. The sequence is presented of a gene on chromosome 10 that lies immediately telomeric of the breakpoints in nine new ALL patients with acquired rearrangements in 10q24. The gene is a novel human homeobox gene and is expressed in leukemic cells from ALL patients with rearrangements in a defined chromosome 10 breakpoint cluster region, but not in other adult tissues or cell lines. This new gene has been designated HOX11. These results strongly support a role for homeobox genes in oncogenesis and may represent the first example of a human cancer in which deregulated expression of an unaltered homeobox gene is involved in tumorigenesis (Dube, 1991).
A common chromosomal abnormality in childhood T-cell acute leukemia is a translocation, t(10;14) (q24;q11), that together with the variant t(7;10)(q35;q24) is present in up to 7% of this tumor type. The gene adjacent to the 10q24 region is transcriptionally activated after translocation to either TCRD (14q11) or TCRB (7q35). It encodes a homeobox gene closely related to the developmentally regulated homeotic genes of flies and mammals. The coding capacity of this activated gene, designated HOX11, is undisturbed in a T-cell line carrying the translocation t(7;10)(q35;q24). Therefore, the HOX11 homeobox gene seems to be involved in T-cell tumorigenesis (Kennedy, 1991).
A translocation involving human chromosome 10, band q24, in a subset of T-cell acute leukemias disrupts a region surrounding the putative oncogene HOX11, which encodes a protein with a homeodomain. The HOX11 protein binds to a specific DNA sequence, it localizes to the cell nucleus, and it transactivates transcription of a reporter gene linked to a cis-regulatory element, suggesting that HOX11 functions in vivo as a positive transcription activator. PCR analysis shows that the HOX11 homeodomain is a member of a distinct class of homeodomains, representatives of which occur in murine and Drosophila genomes. These all contain a threonine residue in place of the more common isoleucine or valine in helix 3 of the homeodomain. HOX11 therefore appears to belong to a family of DNA-binding transactivators of transcription (Dear, 1993).
A cDNA encoding a member of the Tlx/Hox11 family of homeodomain factors from the zebrafish, most closely related to the vertebrate Tlx-1/Hox11 and Tlx-3/Hox11L2 proteins. The gene is expressed in a set of early differentiating neurons that project to a common tract, the lateral longitudinal fascicle. The gene is specifically expressed in spinal cord Rohon Beard neurons, in nucleus of the posterior commissure neurons of the midbrain, in a set of hindbrain neurons that include RoL3 reticulospinal interneurons, and in the trigeminal, statoacoustic, anterior lateral line, glossopharyngeal, and vagal cranial sensory ganglia. Timing of expression of the gene in these neurons correlates with the phase of axonal outgrowth and target innervation. Expression of the gene is also observed in several non-neural tissues, including the pharyngeal arches, budding gill filaments, outgrowing semicircular protrusions in the otic vesicle, and in the pectoral fin buds (Andermann, 2001).
Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Three members of the zebrafish Tlx family are described. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified (Tlx-3a and Tlx-3b), which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia (Langenau, 2002).
Recent evidence suggests that in vertebrates the formation of distinct neuronal cell types is controlled by specific families of homeodomain transcription factors. Furthermore, the expression domains of a number of these genes correlates with functionally integrated neuronal populations. Two members of the divergent T-cell leukemia translocation (HOX11/Tlx) homeobox gene family have been cloned from chick, Tlx-1 and Tlx-3, and they are expressed in differentiating neurons of both the peripheral and central nervous systems. In the peripheral nervous system, Tlx-1 and Tlx-3 are expressed in overlapping domains within the placodally derived components of a number of cranial sensory ganglia. Tlx-3, unlike Tlx-1, is also expressed in neural crest-derived dorsal root and sympathetic ganglia. In the CNS, both genes are expressed in longitudinal columns of neurons at specific dorsoventral levels of the hindbrain. Each column has distinct anterior and/or posterior limits that respect inter-rhombomeric boundaries. Tlx-3 is also expressed in D2 and D3 neurons of the spinal cord. Tlx-1 and Tlx-3 expression patterns within the peripheral and central nervous systems suggest that Tlx proteins may be involved not only in the differentiation and/or survival of specific neuronal populations but also in the establishment of neuronal circuitry. Furthermore, by analogy with the LIM genes, Tlx family members potentially define sensory columns early within the developing hindbrain in a combinatorial manner (Logan, 1998).
HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. The DNA binding specificity of the Hox11 protein was determined by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contains a core TAAGTG motif, consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA (Tang, 1995).
The Ncx gene encodes a homeobox-containing transcription factor that belongs to the Hox11 gene family. Specific Ncx protein binding consensus DNA sequences have been determined. Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (TAAT core) and 5'-CGGTAAGTGG-3' (TAAG core), which coincided with the Hox11 binding sequence. Both Ncx and Hox11 can bind to the TAAT and the TAAG core oligonucleotide in vitro. However, they efficiently transactivate the reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence. Thus, Ncx and Hox11 act as transcriptional activators via their target sequence, 5'-CGGTAATTGG-3' (Shimizu, 2000).
Ectopic expression of the homeobox gene HOX11 is associated with a significant proportion of childhood T-cell acute lymphoblastic leukaemias (T-ALLs). It is hypothesised that one mechanism of gene deregulation involves overcoming the silencing mechanism(s) of gene expression present in normal cells. This study describes a search for trans-acting factors that control transcriptional activity from a distal 5' region of the HOX11 promoter. A region of this promoter has been identified that contributes significantly to HOX11 activation and two distinct regulatory elements are involved. First, a PBX2 Regulatory Element PRE-1048 has been identified which contains a novel DNA-binding sequence and mediates significant activation of the HOX11 gene in K562 cells. This is the first report of a homeobox gene being specifically regulated by PBX2 and the second report of a vertebrate homeobox target gene of a PBX protein. The PREP1 protein was also shown to be part of the PRE-1048-binding complex. The other regulatory element described in this study, RE-1019, contains little sequence conservation to known transcription control elements. It appears that this element is a novel sequence that binds an as yet unidentified factor, mediating significant activation of the HOX11 gene in K562 cells. This is the first detailed report of elements that mediate regulation of the proto-oncogene HOX11 (Brake, 2002).
Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, nested deletions were generated from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5' regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. Several long-range regulatory elements were identified that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models (Brake, 2004).
Many homeobox genes are clustered in a linear array along a chromosome, reflecting their ordered expression along the anterior-posterior axis of the embryo. Expression patterns as well as grafting, ectopic expression and loss-of-function experiments suggest that the Hox genes encode a combinatorial system of positional specification along that axis. In contrast, the function of orphan homeobox genes located at sites outside the four mammalian Hox clusters is less well understood. To assess the functional role of the orphan homeobox gene Hox11, Hox11-deficient mice were generated through gene targeting. Hox11-/- mice have no spleen, but otherwise appear normal. Hox11 is normally expressed in the splenic anlage arising from the splanchnic mesoderm. Hox11-/- embryos have no cellular organization at the site of splenic development but all other splanchnic derivatives develop normally. Hox11 controls the genesis of a single organ, providing new insight into the genetic regulation of morphogenesis (Roberts, 1994).
The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of beta-galactosidase from the recombinant hox11 allele in +/- mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11-/- mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11-/- mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis (Dear, 1995).
The genetic steps governing development of the spleen are largely unknown. Absence of Hox11 in mice results in asplenia, but it is unclear how Hox11 exerts its effect on spleen development. To more precisely define Hox11's role in spleen morphogenesis, the fate of the developing spleen was examined in Hox11(-/-) mice. Perturbation of spleen development begins between dE13 and dE13.5. Cells of the spleen anlage persist past this developmental stage as an unorganized rudiment between the stomach and the pancreas. They fail to proliferate, and haematopoietic cells do not colonize the rudiment. At later stages of embryonic development, the cells can be observed in the mesenchyme of the pancreas, also an expression site of Hox11. In Hox11-/-<-->+/+ chimaeras, spleens are devoid of Hox11(-/-) cells, indicating that the genetic defect is cell autonomous and not due to failure of the organ anlage to attract and retain haematopoietic cells. In chimaeric embryos, Hox11(-/-) cells are initially present in the spleen anlage. However, at dE13, a reorganization of the spleen occurs in the chimaeras and Hox11(-/-) cells are subsequently excluded from the spleen, suggesting that a change in the affinity for one of the spleen cells occurs. These observations demonstrate that spleen development consists of genetically separable steps and that absence of Hox11 arrests spleen development at an early stage. The formation of the spleen primordium before the entry of haematopoietic cells does not require the activity of Hox11. However, subsequent differentiation of spleen precursor cells is dependent on the Hox11 gene (Kanzler, 2001).
Hox11 codes for a homeobox protein that controls genesis of the spleen. Hox 11 is also oncogenic, having been isolated from a chromosomal breakpoint in human T-cell leukemia. Transgenic mice that redirect Hox11 expression to the thymus demonstrate cell-cycle aberrations and progression to malignancy. In order to understand the cell cycle disruptions caused by HOX11 protein, the protein was tested for interaction partners. HOX11 directly interacts with the PP2A catalytic subunit and the related PP1C. The physical interaction domain of HOX11 is outside the homeodomain. PP2A can regulate the cell cycle of Xenopus oocytes by maintaining G2 meiotic arrest preventing activation of maturation-promoting factor. Microinjection of HOX11 into Xenopus oocytes arrested at the G2 phase of the cell cycle promotes progression to M phase. The interaction of HOX11 with PP2a suggests a mechanism by which a homeobox protein can alter the cell cycle (Kawabe, 1997).
HOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. HOX11 immortalizes murine hematopoietic progenitors and induces pre-T-cell tumors in mice after long latency. HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. HOX11 is shown to repress transcription through both TATA-containing and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicates that repressor activity is separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphate-backbone contacts with DNA. The pathologic action of HOX11 involves DNA binding-dependent transcriptional pathways that are distinct from those controlling expression of a chromosomal target gene (Aldh-1). It is concluded that dysregulated expression of a particular set of downstream target genes by DNA binding via the homeodomain is of central importance for leukemia initiation mediated by HOX11 (Owens, 2003).
HOX11 is a proto-oncogene, which is silent in normal mature T-cells, while being aberrantly activated in T-cell acute lymphoblastic leukaemia (T-ALL) by translocations t(10;14)(q24;q11) or t(7;10)(q35;q24). Although many oncogenes are expressed in alternative forms in cancer, thus far, only one form of the human HOX11 transcript has been reported. This study describes the identification of three alternative transcripts of the HOX11 proto-oncogene, expressed in primary T-ALL specimens. Using rapid amplification of cDNA ends (RACE) and targeted RT-PCR, 23 individual cDNA clones have been sequenced to characterise these novel transcripts. Northern hybridisation identified particular novel exons expressed in T-ALL, which are not expressed in normal T-cells. To date, aberrant expression of HOX11 has only been associated with leukaemia. This survey of a range of neuroblastoma and primitive neuroectodermal tumor (PNET) cell lines demonstrates the expression of these novel HOX11 transcripts in tumors of neural origin, while their expression was not detected in normal brain tissues. Strikingly, the dominant transcript in these neural tumor cell lines is more than 1 kb larger than the dominant transcript in T-ALL. These observations, combined with sequence data from several EST clones derived from medulloblastoma cDNA libraries, support a new hypothesis that HOX11 may also function as a neural oncogene or brain tumor marker (Watt, 2003).
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