Human Homeobox Gene Research Articles

A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues.

A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted HB9 protein has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues.

Quantification of biotinylated RNA probes for in situ hybridization using chemiluminescence

For reliable detection of mRNA by non-radioactive in situ hybridization, calibration and standardization of the individual steps involved are essential. We describe a method that allows determination of the size and integrity as well as quantification of biotinylated RNA probes in a single experiment. Serial dilutions of biotinylated RNA probes generated by promoter-mediated in vitro transcription were size-separated by gel electrophoresis in the presence of known amounts of 5'-biotinylated oligomers which served as internal standard. Following immobilization onto nylon membranes and visualization by chemiluminescence, optical densities of probes and internal standards were measured by densitometry and analysed by linear regression. RNA probes complementary to the human homeobox genes HOX-C6, -C8 and -C9 were quantified. Four different 5'-biotinylated oligomers (20, 35, 50 and 75 bases) were tested as internal standards. Concerning the separation of probe and oligomer in the gel, transfer properties and efficiency of binding to the membrane, the oligomer of 35 bases was found to be the best internal standard with highest reproducibility. Comparison of probe concentration by spectrophotometry and the described method showed a good correlation, indicating that our method is a reliable assay for quantitative and qualitative control of biotin-labelled probes.

Identification of the Murine Hox-c12 and Hox-c13 Homeoboxes on Yeast Artificial Chromosomes

The structure of the Antennapedia-class homeobox gene clusters is highly conserved in human and mouse. Of all the human homeobox genes known, only HOX-C12 and HOX-C13 have not also been identified in mouse. These two genes are located at the extreme 5′ end of the human HOX-C cluster and are predicted to be located in a similar position on the murine Hox-c cluster. We have isolated two yeast artificial chromosomes (YACs) containing the murine Hox-c cluster. We designed primers specific to the human HOX-C12 and C13 homeoboxes and used the polymerase chain reaction and sequencing to identify the homeobox of the murine orthologs of these two genes on one of the YACs. Only the YAC containing the 5′ end of the Hox-c cluster contained these sequences. Thus, we have identified two mouse homeobox genes not previously reported and established linkage of these genes to the 5′ end of the mouse Hox-c cluster.

Molecular cloning of the human homeobox gene goosecoid (GSC) and mapping of the gene to human chromosome 14q32.1.

Goosecoid is a homeobox gene first isolated from a Xenopus dorsal lip cDNA library. Homologous genes have been isolated from mouse, zebrafish, and chick. In all species examined, the gene is expressed and plays an important role during the process of gastrulation in early embryonic development. We report here the cloning of the human goosecoid gene (GSC) from a genomic library and the sequence of its encoded protein. The genomic organization and protein sequence of the human gene are highly conserved with respect to those of its Xenopus and mouse counterparts: all three genes consist of three exons, with conserved exon-intron boundaries; the sequence of the homeodomain is 100% conserved in most vertebrates. Using somatic cell hybrid and chromosomal in situ hybridization, the gene was mapped to chromosome 14q32.1.

A Drosophila melanogaster homologue of the T-cell oncogene HOX11 localises to a cluster of homeobox genes.

The human homeobox gene HOX11 has been identified at the site of a chromosomal translocation in a subset of T-cell acute leukaemias. In the mouse genome, the hox11 family consists of at least three related genes, each of which possesses a highly conserved homeobox. To assist in elucidating the roles of this gene, a homologue was studied from Drosophila melanogaster. This gene, 311, shares similar identity to all three murine family members and contains the threonine residue in helix 3 of the homeodomain characteristic of the Hox11 family. It maps to a cluster of NK-homeobox genes which function in muscle development. Gene 311 exhibits a similar temporal pattern of expression to the NK genes in this cluster. Therefore, 311 may constitute part of a homeobox cluster in which the genes are both co-ordinately regulated and functionally related.

Homeobox genes: Potential candidates for the transcriptional control of the transformed and invasive phenotype

The transformation of a cell and the acquisition of the invasive and metastatic phenotype result from the activation of a group of complex cellular processes rather than from the effect of a single gene product. It is likely that the coordination of the multiple genes involved in malignancy is under the control of a few genes that act as master genes or orchestrator genes. The latter probably code for transcription factors that control the genetic program for tumor invasion and metastasis. Homeobox genes are a family of transcription factors that contain a 183 bp highly conserved nucleotide sequence coding for a 61 amino acid domain that binds specifically to DNA. First discovered in Drosphila as genes controlling segmentation and segment identity, homeobox genes have since been identified in many other species including nematodes, frog, mouse and human. There is strong support for the suggestion that homeobox genes play a key role in development and differentiation. In humans, there are 38 homeobox genes organized in four clusters that are localized on chromosomes 2, 7, 12 and 17. The specific functions of each of these genes are generally unknown. Alterations in expression of several homeobox genes have been reported in a variety of malignant lesions, suggesting that they could play a role in the development of cancer. Using reverse transcriptase reaction coupled with polymerase chain reaction and degenerate oligonucleotides corresponding to the 5′ and 3′ ends of the highly conserved homeodomain, we amplified 130 bp cDNA fragments from the human breast cancer cell line MCF7 that were subsequently cloned into pBluescript vector. Sequencing of the clones, resulted in the identification of the homeodomains of four different human homeobox genes: HOXB6, HOXA1, HOXA10 and HOXC6. Further studies should determine the specific role of these four homeobox genes in the development and progression of human breast cancer and potentially determine if they might be good targets for gene therapy.

Characterization of the Homeobox-Containing Gene GH6 Identifies Novel Regions of Homeobox Gene Expression in the Developing Chick Embryo

Homeobox genes are a major group of genes involved in regulating embryogenesis. Here we describe the identification of GH6, a novel chicken homeobox-containing gene and its spatial and temporal expression pattern in the developing chick embryo. Identity comparisons of the GH6 homeodomain suggest that it is closely related to the human homeobox gene H6 , with 93% amino acid conservation. Temporally, GH6 expression is highest between embryonic stages 23 and 26; however, some expression is also detectable as early as stage 13. In situ hybridization of stage 23 embryos indicates that GH6 expression occurs at high levels in discrete craniofacial regions including the second branchial arch, the neural retina, the lens epithelium, the optic nerve, and the infundibulum. GH6 expression was also seen in the developing ventricular myocardium, representing the first report of homeobox gene expression in the developing ventricle. GH6 is also expressed in sensory spinal and cranial ganglia, suggesting that GH6 plays several roles not only in the development of craniofacial structures such as the eye and ear, but also in formation of functionally defined ganglia and myocardial structures.

Molecular Characterisation of Three Murine HOX11-Related Homeobox Genes, Tlx-1, -2, and -3, and Restricted Expression of Tlx-1 during Embryogenesis: (murine/homeobox/cloning/motif/expression).

Elevated expression of the human homeobox gene HOX11 has been implicated in T cell acute lymphoblastic leukemia. We report here the isolation and characterisation of three murine genes, Tlx-1, -2, and -3, which are related to HOX11 and constitute a new class of homeobox genes. Sequence analysis indicates that Tlx-1 is a murine homolog of HOX11. The expression during development of Tlx-1 was studied by in situ hybridization. Tlx-1 expression was found in the first branchial arch, the pancreas, the hindbrain and the epidermis of the ear of the developing embryos. Localized expression of Tlx-1 in these developing structures suggests that it might play a specific role during post-implantation development and its aberrant expression might be involved in oncogenesis. The homeodomains of Tlx-2 and -3 are found to be 75% and 80% identical to that of Tlx-1, respectively. In addition to the homeodomain, the three Tlx proteins also share four conserved motifs (TH1-TH4). Interestingly, these motifs are also found in other developmental control genes and transcription factors, suggesting a functional role in transcriptional regulation.

Characterization and developmental expression of Tlx-1, the murine homolog of HOX11

HOX11, a human homeobox gene with putative oncogenic potential, was originally discovered at the chromosome 10 breakpoint in T-cell acute lymphoblastic leukemias bearing the chromosomal translocation t(10;14)(q24;q11). To provide insight into the possible roles of this gene in development, we isolated and characterized its murine homolog, Tlx-1, and examined its profile of expression. Tlx-1 transcripts are first detected at E8.5 in the surface ectoderm and central mesenchyme of the first branchial arch. This expression subsequently extends to the 2nd, 3rd, and 4th branchial arches, as well as the presumptive pharynx, as these structures develop. Between E12.5 and E15.5, the profile of Tlx-1 expression becomes more complex; expression is observed in the developing pancreas and salivary glands, as well as in several components of the nervous system, including the trigeminal, glossopharyngeal and vestibulocochlear ganglia, the spinal cord, and the curvature of the pons-medulla. In addition, expression is seen in the pinna and external auditory meatus of the outer ear, the tooth primordia, and specific cell populations of the mandible and tongue. These complex patterns of expression are consistent with multiple and varied roles for Tlx-1 in development and suggest that Tlx-1 marks, amongst other cell populations, structures derived from cranial neural crest cells and migratory paraxial mesoderm that arise at corresponding levels along the rostral-caudal axis of the developing embryo.

A human homeobox gene, HB24, inhibits development of CD4+ T cells and impairs thymic involution in transgenic mice.

The HB24 gene encodes a diverged human homeodomain-containing protein known to be expressed in hematopoietic progenitors and activated lymphocytes. We have generated transgenic mice that express HB24 under the control of the T cell receptor beta chain promoter/enhancer. Analysis of T cells and thymocytes from the transgenic mice revealed a marked increase in activated cells as assessed by cell size profiles and interleukin-2 receptor expression. Within the thymus these changes were most pronounced in the CD4+CD8- subset. Strikingly, the normal development of CD4+ T cells in the transgenic mice was impaired. Single positive CD4 cells were reduced 35% in the thymus, and CD4+ T cells were reduced 90% in the spleen and lymph nodes compared to the controls. Similar findings were found both in young mice (6 weeks old) and in more elderly mice (1 y old). However, the thymuses of the elderly mice failed to undergo normal involution. Sera from HB24 transgenic mice had levels of IgG1 10-100-fold lower than sera from matched controls, most likely as a consequence of the decrease in CD4+ T cells. These transgenic mice provide a useful model for studying the role of HB24 in lymphocyte activation as well as for understanding the effects of abnormal T cell activation on thymic and T cell development.

Linkage Localization of TGFB2 and the Human Homeobox Gene HLX1 to Chromosome 1q

We have identified genetic variation within two human genes, transforming growth factor-β2 (TGFB2) and the homeobox gene HB24 (HLX1). Reported here are four human RFLPs and SSCPs for TGFB2 in humans and gorillas. In addition, we describe an RFLP and a SSCP for HLX1. We propose that HLX1 is the human homologue of the mouse homeobox gene Hlx based on extensive sequence homology between the genes and the close proximity of both genes to TGFB2 in their respective species. We also report the chromosomal localization of HLX1 to the long arm of human chromosome 1. Finally, utilizing the polymorphisms described for TGFB2 and HLX1, we have been able to localize these genes within a framework map of the distal long arm of chromosome 1 and to study the linkage relationship between these two genes. Pairwise linkage analysis shows that these two genes are linked, with a recombination fraction of 3.1% and a lod score of 14.49.

Potential roles for two human homeodomain containing proteins in the proliferation and differentiation of human hematopoietic progenitors.

Two human homeobox genes, HB9 and HLX, are expressed in hematopoietic progenitors and activated lymphocytes. They are implicated in the proliferation of hematopoietic progenitors in response to growth factors and the differentiation of hematopoietic progenitors to mature cell lineages. RNAs from bone marrow cells of patients with acute myeloid or lymphocytic leukemia have high levels of these two genes while similar RNAs from patients with chronic lymphocytic or myeloid leukemias have nearly normal levels. While the significance of these two genes in leukemogenesis is unknown, they are likely to regulate gene transcription during hematopoiesis and their dysregulation may have dire consequences for hematopoietic cells.

Expression of homeobox-containing genes in primary and metastatic colorectal cancer.

Homeobox genes are a network of genes encoding nuclear proteins functioning as transcriptional regulators. Human and murine homeobox genes of the HOX family are organised in four clusters on different chromosomes. Gene order within each cluster is highly conserved, perhaps in direct relation to their expression. Homeobox genes have recently been involved in normal development and oncogenesis. We have analysed HOX gene expression in normal human colon and in primary and metastatic colorectal carcinomas. The majority of HOX genes are active in normal adult colon and their overall expression pattern is characteristic of this organ. Furthermore, the expression of some HOX genes is identical in normal and neoplastic colon indicating that these genes may exert an organ-specific function. In contrast, other HOX genes exhibit altered expression in primary colon cancers and their hepatic metastases which may suggest an association with colon cancer progression.

Two diverged human homeobox genes involved in the differentiation of human hematopoietic progenitors map to chromosome 1, bands q41-42.1.

Proteins encoded by homeobox containing genes are sequence-specific DNA binding proteins implicated in the control of gene expression in both developing and adult tissues. Two recently characterized human homeobox genes, HB9 and HB24, are highly expressed in CD34-positive marrow cells but not in CD34-depleted marrow cells. Their expression is readily down-regulated during the differentiation of hematopoietic progenitors to specific cell lineages. In this study, genomic DNA fragments isolated with HB9 (3 kb) and HB24 (6 kb) cDNAs were used to map their chromosomal location by fluorescence in situ hybridization. Both HB9 and HB24 DNA probes gave specific hybridization signals on chromosome 1. The hybridization loci were identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by cohybridization in situ with an Alu probe (R-like banding) and by DAPI staining (G-like). The results demonstrate that the loci of the HB24 and HB9 genes are within bands 1q41-q42.1. A cohybridization experiment utilizing both probes with two-color fluorescence imaging could not resolve separate loci for the two genes.

Cloning and sequencing of the human homeobox gene HOX4A

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60 c-src , Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60 c-src . A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.

Expression of selected human HOX-2 genes in B/T acute lymphoid leukemia and interleukin-2/interleukin-1 beta-stimulated natural killer lymphocytes

Although the key role of human homeobox (HOX) genes in development is well established, their function in adult cells is still under scrutiny. We have analyzed, in normal adult blood cell subpopulations, acute lymphoid leukemia (ALL) cells lines, and primary blasts, the RNA expression of all HOX-2 cluster genes (5'-2.5, 2.4, 2.3, 2.2, 2.1, 2.6, 2.7, 2.8, 2.9, 3') and nine genes in the HOX-1, -3, and -4 cluster by Northern blotting, RNAse protection, and/or reverse transcriptase polymerase chain reaction (RT-PCR). The analyzed HOX-1, -3, and -4 genes were never expressed in all tested cell populations. Natural killer (NK) cells activated in interleukin-2 (IL-2)/IL-1 beta-treated cultures exhibit a gradually increasing, abundant expression of three HOX-2 genes (2.2, 2.6, 2.8), while three other genes (2.3, 2.1, 2.7) are expressed at a lower level at late culture times. However, no HOX-2 gene is expressed in quiescent lymphocytes (NK, B and T [T-cell receptor (TCR) alpha/beta, gamma/delta lymphocytes, thymocytes] cells), granulocytes, and monocytes. In B- and T-ALL cell lines, HOX-2 genes are expressed according to different patterns: (1) widespread transcription (seven of nine genes, including 2.3 and 2.6) in the Peer line bearing the TCR gamma/delta; (2) expression of 2.5, 2.2, and 2.6 in the SEZ 627 line, which derives from an HTLV-1+ T-helper leukemia; (3) transcription of 2.3 and 2.6 in both the T-ALL CEM line and four B-ALL lines (interestingly, CALLA- B-ALL lines are constantly 2.3/2.6 RNA+); (4) no HOX-2 gene expression was detected in one T- and two B-ALL lines. Primary blasts from five T- and five pre-B-ALL showed selective expression of one or more HOX-2 genes, namely 2.5, 2.2, 2.6, and 2.7. Our data are compatible with the hypothesis that selected HOX-2 genes play a role in the IL-2/IL-1 beta-induced activation and/or proliferation of normal NK lymphocytes and possibly in the oncogenetic process of some T- and B-ALL.

Expression of Selected Human HOX-2 Genes in B/T Acute Lymphoid Leukemia and Interleukin-2/Interleukin-l β-Stimulated Natural Killer Lymphocytes

Abstract Although the key role of human homeobox (HOX) genes in development is well established, their function in adult cells is still under scrutiny. We have analyzed, in normal adult blood cell subpopulations, acute lymphoid leukemia (ALL) cells lines, and primary blasts, the RNA expression of all HOX-2 cluster genes (5′-2.5, 2.4, 2.3, 2.2, 2.1, 2.6, 2.7, 2.8, 2.9, 3′) and nine genes in the HOX-1, -3, and -4 cluster by Northern blotting, RNAse protection, and/or reverse transcriptase polymerase chain reaction (RT-PCR). The analyzed HOX-1, -3, and -4 genes were never expressed in all tested cell populations. Natural killer (NK) cells activated in interleukin-2 (IL-2)/IL-1 beta-treated cultures exhibit a gradually increasing, abundant expression of three HOX-2 genes (2.2, 2.6, 2.8), while three other genes (2.3, 2.1, 2.7) are expressed at a lower level at late culture times. However, no HOX-2 gene is expressed in quiescent lymphocytes (NK, B and T [T-cell receptor (TCR) alpha/beta, gamma/delta lymphocytes, thymocytes] cells), granulocytes, and monocytes. In B- and T-ALL cell lines, HOX-2 genes are expressed according to different patterns: (1) widespread transcription (seven of nine genes, including 2.3 and 2.6) in the Peer line bearing the TCR gamma/delta; (2) expression of 2.5, 2.2, and 2.6 in the SEZ 627 line, which derives from an HTLV-1+ T-helper leukemia; (3) transcription of 2.3 and 2.6 in both the T-ALL CEM line and four B- ALL lines (interestingly, CALLA- B-ALL lines are constantly 2.3/2.6 RNA+); (4) no HOX-2 gene expression was detected in one T- and two B- ALL lines. Primary blasts from five T- and five pre-B-ALL showed selective expression of one or more HOX-2 genes, namely 2.5, 2.2, 2.6, and 2.7. Our data are compatible with the hypothesis that selected HOX- 2 genes play a role in the IL-2/IL-1 beta-induced activation and/or proliferation of normal NK lymphocytes and possibly in the oncogenetic process of some T- and B-ALL.

Stable expression of HB24, a diverged human homeobox gene, in T lymphocytes induces genes involved in T cell activation and growth.

A diverged homeobox gene, HB24, which is known to be induced following lymphocyte activation, was introduced into Jurkat T cells under the control of a constitutive promoter. Stable transfectants of HB24 were established that expressed high levels of HB24 mRNA and possessed an altered phenotype suggestive of activated T cells. A number of genes known to be induced following T cell activation and associated with cell growth were increased in the transfectants, including c-fos, c-myc, c-myb, HLA-DR, lck, NF-kappa B, interleukin-2 and interleukin-2 receptor alpha (IL-2R alpha). Analysis of IL-2R alpha expression by transient transfection of IL-2R alpha promoter constructs into the HB24 transfectants revealed constitutive expression (about 60% of phytohemagglutinin- and phorbol ester-activated Jurkat cells) that was dependent on the kappa B site in the IL-2R alpha promoter. Furthermore, as a consequence of the increased HB24 mRNA levels, the Jurkat HB24 transfectants proliferated more rapidly than control cell lines. Thus, stable expression of HB24 confers an activation phenotype on a human T cell line, implicating this gene as an important transcriptional factor during T cell activation and growth.

The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and HOX homeoproteins.

We studied the structure, regulation and expression of HOX3D, a human homeobox gene located in the HOX3 cluster on chromosome 12. HOX3D is developmentally regulated during embryogenesis and is activated by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. Transfection of HOX3D upstream genomic sequences linked to a reporter gene allowed the functional definition of its promoter, containing a canonical TATA element. This promoter directs the expression of the reporter gene in EC cells after induction with RA, and binds RA-induced nuclear factor(s) through a conserved palindromic sequence located approximately 100 bp upstream of the transcription start site. The HOX3D promoter is transactivated in both human and murine cells when cotransfected with vectors expressing the protein product of the upstream gene HOX3C and the paralogs of further upstream genes in the HOX4 cluster (i.e. HOX4D, HOX4C and the murine Hox 4.3). The HOX3D protein, and those encoded by the downstream gene HOX3E and its paralog HOX4B are instead inactive. HOX4C and HOX4D proteins synthesized in bacteria bind to the same conserved sequence located around position -120, as well as to the TATA box and immediately upstream and downstream nucleotides. These data provide evidence that cross-regulatory interactions between mammalian homeogenes take place in cultured cells, thus raising the possibility that a regulatory network may exist in vivo. The sequences on the HOX3D promoter involved in cross-regulation are different from those binding nuclear factors induced by RA.

A novel human homeobox gene lies at the chromosome 10 breakpoint in lymphoid neoplasias with chromosomal translocation t(10;14)

The translocation t(10;14)(q24;q11) is an acquired change seen in 4% to 7% of T-cell acute lymphoblastic leukemias (T-ALL). We previously demonstrated that 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. We now present the sequence 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. Our 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.