Stem Cells, Vol. 14, No. 3, 281-291,
May 1996
© 1996 AlphaMed Press
The Role of HOX Homeobox Genes in Normal and Leukemic Hematopoiesis
H. Jeffrey Lawrencea,
Guy Sauvageaub,
R. Keith Humphriesc,
Corey Largmana
a Veterans Affairs Medical Center, University of California, Department of Medicine, San Francisco, California, USA;
b Clinical Research Institute of Montreal, Montreal, Quebec, Canada;
c Terry Fox Laboratory, British Columbia Cancer Agency, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Key Words. Homeobox genes • Hematopoiesis • Transcription factors • Transgenic mice • Stem cells • Leukemia
Dr. Corey Largman, Hematopoiesis Research (151H), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA.
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Abstract
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A sizable amount of new data points to a role for the HOX family of homeobox genes in hematopoiesis. Recent studies have demonstrated that HOXA and HOXB genes are expressed in human CD34+ cells, and are downregulated as cells leave the CD34+ compartment. In addition, expression of certain genes, including HOXB3 and HOXB4, is largely restricted to the long-term culture-initiating cell enriched pool, containing the putative stem cell population. Studies have also shown that HOX genes appear to be important for normal T lymphocyte and activated natural killer cell function. Overexpression of Hox-b4 in transplanted murine marrow cells results in a dramatic expansion of stem cells, while maintaining normal peripheral blood counts. In contrast, overexpression of Hox-a10 resulted in expansion of progenitor pools, accompanied by unique changes in the differentiation patterns of committed progenitors. Overexpression of Hox-a10 or Hox-b8 led to the development of myeloid leukemias, while animals transfected with marrow cells overexpressing Hox-b4 do not appear to develop malignancies. Blockade of HOX gene function using antisense oligonucleotides has revealed that several HOX genes appear to influence either myeloid or erythroid colony formation. Mice homozygous for a targeted disruption of the Hox-a9 gene show reduced numbers of granulocytes and lymphocytes, smaller spleens and thymuses, and reduced numbers of committed progenitors. These studies demonstrate that HOX homeobox genes play a role in both the early stem cell function as well as in later stages of hematopoietic differentiation, and that perturbations of HOX gene expression can be leukemogenic.
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Introduction
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The term homeobox gene arose from an earlier genetic term, the so-called homeotic mutation, a mutation in which the identity of one body segment is converted to that of another. Such mutations were first described in Drosophila early in this century [1], and are exemplified by a mutation in which flies have legs in place of their antennae. The cloning of the gene, called Antennapedia, involved in this mutation over a decade ago represented the identification of the first homeobox gene [2 ,3]. Since that time, a large body of data has emerged on the master regulatory role of homeobox genes in embryogenesis, both in insects and in vertebrates [4– 6 ]. These genes contain a highly conserved 183-nt homeobox encoding a DNA-binding motif, referred to as the homeodomain. Homeodomain proteins are transcription factors which play a profound role in specifying relative positions and tissue fate in the embryo, particularly along various body axes, including the A-P axis and the axes of the developing limb bud.
Mammalian homeobox genes can be divided into two broad categories: a family of 38 genes which are related to the Antennapedia gene from Drosophila, and other more distantly related genes. The Antennapedia-like genes are referred to as the HOX family and are organized into four clusters as the result of an ancestral quadruplication of a single cluster of genes [7 ]. The four HOX clusters each contain 8-11 genes and are localized to different chromosomes [8 ]. Individual genes in different HOX clusters can be aligned with each other on the basis of sequence homology within their homeobox regions, and somewhat surprisingly, with homeobox genes of the Drosophila HOM-C cluster [9 ], indicating conservation of these ordered gene clusters over a broad span of evolutionary time. These so-called paralogs (e.g., HOXA4, HOXB4, HOXC4 and HOXD4) also show varying degrees of homology outside the homeobox.
Figure 1
shows the organization and standardized names of the 38 human HOX genes [10]. The figure also includes the old nomenclature since they are used in many of the referenced articles. Human genes are referred to in upper case (e.g., HOXA4) while murine genes are referred to in lower case (e.g., Hox-a4); in this article, the proteins from both species are in lower case and non-italicized (e.g., Hox-a4). The second category of divergent homeobox genes consists of a large number of genes scattered throughout the genome. Several of these genes were discovered on the basis of their involvement with leukemic chromosomal rearrangements [11,12]. In this review, we will focus on recent evidence of a role for HOX genes in hematopoietic stem cell growth and differentiation.
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Figure 1. Four mammalianHOX clusters with old and new nomenclature. [Reproduced from Cell, 1992;71:551-553, by copyright permission of Cell Press] [10].
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Individual HOX genes are expressed in precise temporal and spatial patterns in the embryo. Genes within a cluster show overlapping zones of expression from head to tail along the spinal axis, with progressively more anterior rostral boundaries of expression as one moves from 5' to 3' in the cluster [9, 13 ]. Thus there is colinearity between the head-to-tail patterns of HOX gene expression and the order of individual HOX genes in a cluster. A similar overlapping pattern of HOX gene expression develops later along the proximal-distal axis of the developing limb bud. These observations suggest that HOX genes are involved with the specification of tissue identity along various body axes. The profound influence of Hox proteins on vertebrate morphogenesis is best evidenced by the often dramatic regional alterations of body pattern particularly in the axial skeleton and the limbs, seen in transgenic mice bearing targeted disruptions or overexpression vectors for specific homeobox genes [14]. Despite the explosion of evidence linking HOX genes to embryogenesis, very little is known about the target genes or the precise mechanism of action for any Hox protein, although as will be discussed below, gene targets may include those encoding adhesion molecules, extracellular matrix proteins, growth factors and perhaps globin.
The establishment of ordered domains of HOX gene expression in the embryo is thought to be controlled by gradients of various putative morphogens, such as retinoic acid, and in embryonic carcinoma cells, HOX genes show an ordered and dose-related activation by treatment with retinoic acid that is also correlated with the 3' to 5' order within a given HOX cluster [15]. Interestingly, inactivation of expression of a 3' gene by treatment with an antisense oligonucleotide (ASO) blocks the retinoic acid-induced activation of more 5' genes [16]. There is circumstantial evidence that peptide growth factors such as transforming growth factor ß (TGF-ß), activin and fibroblast growth factor may also regulate HOX gene activation [17].
The transcriptional regulation of HOX genes themselves is poorly understood. There is evidence for complex auto- and cross-regulatory control of HOX genes by their protein products [18–20]. The activation of HOX genes by retinoids indicates a possible role for retinoic acid receptors in the regulation of HOX gene transcription, and there appear to be retinoic acid response elements 3' of the HOXA1 gene and 5' of HOXD4 [21,22]. Finally in Drosophila the protein encoded by the trithorax gene appears to be a transcription factor which positively regulates homeobox genes in the fly and is essential for maintenance of homeobox gene expression during embryogenesis. The human homolog of trithorax is the MLL (also referred to as ALL-1 or HRX) gene on chromosome 11q23 [23–25], and it appears to exert a similar positive regulatory effect on HOX genes. In very recent work, the MLL gene has been interrupted in mice by homologous recombination. In mice heterozygous for the mutant allele, the pattern of HOX genes along the A-P axis is shifted posteriorly, while in homozygous animals the HOX genes are not expressed at all [26].
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HOX Gene Expression in Hematopoietic Cell Lines
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The initial reports of HOX gene expression in blood cells involved the demonstration of expression in immortalized cell lines of both human [27–32] and murine origin [33,34]. While some HOX genes show broad expression in cells of different phenotypes, several appear to have lineage-restricted patterns of activation. Eight of the nine members of the human HOXB (formerly HOX 2) cluster are expressed primarily in cell lines with erythroid potential [27–30]. Several of the HOXB genes, including HOXB4 and HOXB7, are also expressed in a range of T and B cell lines [35–38]. Certain HOXC genes have been reported to be selectively active in erythroid cell lines [39]. The HOX genes with the greatest apparent specificity of expression are HOXA10, which is expressed predominantly in myelomonocytic cell lines [40] and HOXC4, which is restricted to lines with lymphoid character [31]. Expression of the HOXD genes, with rare exception [41], has not been detected in any of the leukemic cell lines surveyed.
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Expression of HOX Genes in Primitive Hematopoietic Cells from Normal Marrow
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These results were extended to the demonstration of expression of a number of HOX genes including HOXB6, HOXB2 and HOXA10 in unfractionated normal human marrow by RNase protection, indicating that HOX gene expression was not simply a hallmark of transformed cells [29,31,40]. To determine the stages at which HOX genes are activated during blood cell differentiation, a number of laboratories have used reverse transcription-polymerase chain reaction (RT-PCR) to detect HOX gene expression in small numbers of purified human hematopoietic progenitors [42–45]. Taken together, these studies demonstrate that there is a marked trend for expression of HOXA > HOXB >> HOXC, with HOXD genes being silent in hematopoietic progenitors.
In the most thorough study to date, Sauvageau et al. demonstrated expression of a number of HOXA and HOXB genes in highly enriched subpopulations of human CD34+ marrow cells, including fractions containing the putative stem cell, as defined by long-term culture-initiating cell (LTC-IC) assay. HOXA genes are expressed at higher levels than HOXB genes, and genes in the 3' portion of the HOXA and HOXB clusters are downregulated as CD34+ cells progress to the stage of committed erythroid and myeloid progenitors, while more 5' genes remain active into the committed progenitor stage and are inactivated as cells leave the CD34+ compartment. For example, the HOXA10 gene is expressed at high levels throughout the CD34+ compartment and downregulated in more mature populations, while HOXB3 expression is largely restricted to the earliest progenitor populations, containing the LTC-IC fraction [43]. Giampaolo and co-workers have confirmed the restriction of HOXB3 expression to the earliest human progenitors, and have observed a wave of HOXB gene expression during in vitro erythroid or granulocytic maturation, with progressively more 5' genes being expressed at later times. Expression of the extreme 5' end of the HOXB cluster was not detected in these studies [44].
Much less data exist on the expression patterns of Hox genes in murine cells, so it has not yet been ascertained that the expression patterns seen in human marrow cells are reiterated in other mammals. A number of Hox-a, -b and -c genes are expressed in the murine yolk sac, the first mammalian hematopoietic organ [46]. In particular, very recent work has indicated that the murine Hox-b6 gene is expressed in embryonic yolk sac, fetal liver and in adult colony-forming units-erythroid (CFU-E), but not in pluripotent stem cells [47]. It is interesting to note that HOXB6, unlike neighboring HOXB genes, is not detectably expressed in pluripotent human marrow cells [43], and that overexpression of HOXB6 in an erythroid cell line does perturb erythropoiesis (see below). Taken together, these observations suggest that this gene may have a very specific role in erythropoiesis. Thus the mouse model should prove particularly useful for tracking HOX gene expression during early development [48,49].
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HOX Gene Expression in Lymphoid Cells
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A number of studies have reported HOX gene expression in normal lymphoid cells. The HOXC4 gene appears to be silent in CD34+ progenitor cells, but is activated during early to intermediate stages of both T and B lymphoid development [31]. Several of the HOXB genes are expressed in activated natural killer (NK) cells, but genes of the HOXA, HOXC and HOXD loci could not be detected in T cell populations either before or following activation [37]. Members of the HOXB locus are not expressed in resting T lymphocytes, but are coordinately activated by phytohemagglutinin stimulation [50].
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HOX Gene Expression in Primary Leukemic Cells
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A number of studies have reported the expression of specific HOX genes in certain types of human leukemia. For example, HOXC4 expression is limited to lymphoid leukemias [31,51]. Conversely, the HOXA10 gene appears to be strongly expressed in myeloid leukemias (with the curious exception of acute promyelocytic leukemia), and silent in lymphoid leukemias [51,52]. A block of the HOXB genes is also expressed in acute myeloid leukemias, but is switched off in chronic myelogenous leukemia [51]. These findings suggest that patterns of HOX gene expression could have diagnostic utility, a premise currently being tested in childhood leukemias by the Children's Cancer Study Group [Dr. I Bernstein, personal communication].
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Inappropriate Expression ofHOX Genes in Myeloid Leukemias
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As noted above, an early study showed that a murine leukemic cell line contained an insertion of an intracisternal A particle into the Hox-b8 gene, resulting in constitutive gene expression [53]. Very recently, two groups have obtained the first data indicating a role for Hox-A9 and Hox-A7 in human leukemias [54,55]. In these studies, Hox-A9 has been identified as part of a fusion protein with a nucleoporin protein, NUP98, in t(7,11)(p15,p15) translocations associated with acute myeloid leukemias [54, 55]. The DNA-binding homeodomain is preserved in these fusion proteins, suggesting that the NUP98 protein fragment might either misdirect HOX-A9 protein function to an inappropriate location, or act as an inappropriate activation or repression motif in the context of Hox-A9 mediated gene regulation. In addition, Hox-a9 and Hox-a7 have recently been shown to be activated by proviral integration in the BXH-2 mouse model of myeloid leukemia [56]. It is of interest that the resulting high level expression of the homeobox genes is correlated with the simultaneous proviral insertion into the novel, Pbx-related, Meis gene [57]. Given the recent observations that Hox proteins appear to cooperatively bind DNA with Pbx proteins (see below), the role of Meis-mediated Hox protein binding in both normal hematopoiesis and in leukemias appears to be a fertile area for investigation.
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Modulation of HOX Gene Expression Can Alter the Phenotype of Hematopoietic Cell Lines
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Human leukemic cell lines transfected with vectors allowing high level expression of sense and antisense mRNA of the HOXB6 gene showed shifts in a number of erythroid features, indicating that HOX genes can modulate blood cell phenotype [58]. In addition, it has been observed that HOXB7 is transiently expressed in HL60 during tumor-promoting activity-induced monocytic differentiation, and that inhibition of HOXB7 expression blocks this induction [59]. Finally, Blatt et al. found that overexpression of Hox-b8 (formerly HOX 2.4) inhibits the myeloid differentiation of an inducible murine cell line [60].
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Modulation of HOX Gene Expression Effects Normal Hematopoiesis
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Perhaps the most dramatic evidence for a putative role for HOX genes in regulation of hematopoiesis has been obtained using the techniques of retroviral mediated transfection of Hox genes into murine marrow and generation of mice carrying homologous deletions of Hox genes. Since earlier studies had shown that a murine leukemic cell line contained an insertion of an intracisternal A particle into the Hox-b8 gene, resulted in constitutive gene expression [53], Perkins et al. investigated the effects of overexpressing the Hox-b8 gene in murine bone marrow cells. Overexpression of the Hox-b8 (formerly HOX 2.4) gene in normal mouse marrow resulted in cells which were greatly enhanced in their capacity to form interleukin 3-dependent cell lines as well as an apparent increase in self-renewal of early clonogenic cells, an expansion of progenitor cells, and a propensity to evolve to leukemia over time [61].
More recently, Sauvageau et al. demonstrated that transfection of normal murine bone marrow with a vector overexpressing the Hox-b4 gene resulted in a dramatic increase in the most primitive cell populations, under conditions in which peripheral blood counts remained normal and the recipient mice did not develop malignancies [62]. In these studies, serial transplantation revealed a greatly enhanced ability of Hox-b4-transduced marrow cells to regenerate the most primitive hematopoietic stem cell compartment, resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with animals receiving marrow transfected with a control vector expressing neomycin resistance.
In contrast to the finding of Hox-b4, overexpression of Hox-a10 not only resulted in increased hematopoietic progenitor pools, but also resulted in a dramatic increase in the number of megakaryocytic and primitive blast colonies [63]. In addition, Hox-a10-overproducing marrow cells did not contribute to B cell pools in recipient animals, suggesting that downregulation of the Hox-a10 gene is a prerequisite for B cell maturation. Finally, in a manner reminiscent of Hox-b8 transfected recipients, animals transfected with Hox-a10 are extremely susceptible to a myeloproliferative disorder which resembles chronic myelogenous leukemia. Transplantation of primary marrows from Hox-a10 animals leads to a fulminant acute myeloid leukemia in secondary recipients.
Although the overexpression of Hox genes in bone marrow cells demonstrates dramatic effects which appear to be unique to individual genes, these studies must be interpreted with caution. It is unclear whether the phenotypes resulting from increased abundance of these potent transcription factors are illustrating some aspects of their normal function, or are merely reflective of abnormally high expression levels. In this regard, experimental approaches which ablate the normal expression of HOX genes may prove to be more physiologically relevant. Two approaches have been taken to reduce the levels of HOX gene expression in blood cells: the use of antisense oligonucleotides (ASOs) to diminish Hox gene function in vivo, and targeted disruption of genes by homologous recombination in embryonic stem cells.
Several groups have shown that downregulation of specific HOXB and HOXC genes in marrow cells grown under colony-forming conditions by ASOs appears to inhibit normal human erythroid colony formation [64, 65], whereas treatment with ASOs against HOXA5 simultaneously inhibits myeloid colony formation and enhances erythroid progenitor growth [66]. A potential drawback to this approach is the uncertainty about the level of suppression achieved by ASO treatment; in many cases where levels of mRNA are measured, the reductions are on the order of 50%. Another concern is specificity and whether the ASOs used might be affecting transcripts of other genes.
More recently, investigators have begun to examine the hematological effects of targeted interruption of Hox genes in transgenic mice using the methodology of homologous recombination in embryonic stem cells [67]. Mice lacking a functional Hox-a9 gene have defects in both granulocytic and lymphocytic pathways [68]. These mutant animals, who are otherwise healthy and fertile, have reduced numbers of peripheral blood granuloyctes and lymphocytes, smaller spleens and thymuses, reduced numbers of myeloid and pre-B cell progenitors, but no significant change in pluripotent progenitors as measured by CFU-spleen and LTC-IC assays. These findings demonstrate a physiologic role for Hox genes in normal blood cell formation, and suggest that, in the case of Hox-a9, loss of function affects hematopoiesis primarily at the level of the committed progenitor and not earlier stages. It should be extremely informative to examine the hematopoietic systems of mice bearing targeted interruptions of other Hox genes that are also normally expressed in blood cells.
Very intriguing, albeit indirect, evidence for a physiologic role of Hox genes in hematopoiesis comes from the preliminary findings of animals bearing a targeted disruption of the MLL gene [26]. As described above, this gene is the homolog of the Drosophila trithorax gene, a positive regulator of homeobox genes in the fly. The MLL gene was initially discovered because of its involvement in translocations found in infantile leukemias and in leukemias induced by topoisomerase II inhibitors, and many of these leukemias show promiscuous expression of both myeloid and leukemia features. The animals with disrupted MLL genes have, in addition to abnormalities in Hox gene expression (described above), a number of incompletely characterized defects in hematopoiesis involving both erythroid and B lymphoid cells [26], and a more thorough description of the blood cell phenotype in the animals should be very informative.
An attractive model for examining Hox gene function in embryonic and fetal hematopoiesis has been the in vitro culture system of embryonic stem (ES) cells. Under the appropriate culture conditions normal ES cell lines will differentiate into embryoid bodies, which reiterate many of the normal early programs of development, including hematopoiesis, which can be monitored by tracking the appearance of clonogenic progenitors and by measuring blood-specific gene transcripts [69, 70]. ES cells can be engineered to carry expression vectors of specific genes for gain-of-function experiments and to carry targeted interruptions for loss of function [71]. An advantage of the ES system is that blood development can still be studied in vitro even if the mutation under study is lethal to a normal embryo grown in vivo. This system has been used to study the impact of overexpression of Hox-b4, using a retroviral expression vector, on in vitro hematopoietic development in the embryoid body [72]. In these experiments overexpression of Hox-b4 resulted in a significant increase in the number of mixed erythroid/myeloid and pure erythroid colonies without altering the generation of pure myeloid colonies. The effect on red cell development was primarily on definitive and not primitive erythropoiesis, since adult ß-globin was significantly increased with no change in levels of GATA-1 or embryonic globin (ßH-1). It is intriguing to note that, in earlier studies in cell lines, HOXB4 gene expression was noted in erythroid but not myeloid leukemic cell lines.
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Target Genes for Homeodomain Proteins
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The genomic binding sites for Hox proteins in any tissue of any animal species are largely unknown. Genetic experiments in Drosophila suggest that decapentaplegic (dpp) and wingless genes (wg), both homologs of mammalian peptide growth factors, are targets of homeobox proteins [73]. Among the few targets identified for Hox protein binding are several molecules which are involved in cell-cell interactions, including the connectin gene [74] which encodes a novel cell adhesion molecule involved in the innervation of muscle [75]; the scabrous (sca) gene [76], which appears to produce a secreted protein involved in cellular communication during neurogenesis; and a homolog of the Drosophila tumor-suppressor gene l(2)gl [77], which appears to be a cell adhesion molecule with homology to the cadherin family [78]. Other investigators have demonstrated the binding and transcriptional modulation of the 5' regulatory region of the NCAM gene by Hox-b8, Hox-b9 and Hox-c6 [79–82], and the binding of Hox-d9 to the E-cadherin enhancer.
Given the evidence for a role of homeobox genes in the regulation of adhesion molecules, it is important to note that blood cells express a wide array of such molecules, including the integrin family, the ICAMS and the selectins [83–86]. Various cell surface proteins are thought to mediate interactions between hematopoietic progenitors and stromal cells within the marrow which regulate early differentiation events. In addition, cell surface proteins subserve a large array of mature blood cell functions, including the interaction of leukocytes with vascular endothelium, with extracellular matrix and with each other. The evidence cited above suggests that Hox gene targets may include genes encoding adhesion molecules and peptide growth factors. Although no one has demonstrated direct regulation of blood-specific adhesion molecules or cytokines by Hox proteins, one group has shown that overexpression of HOXD3 in human erythroleukemia cells results in increased adherence of the cells to a variety of substrates and an increased level of integrin ß3 mRNA [41].
Several studies suggest a potential role for Hox proteins in the regulation of globin expression [87, 88]. One group identified a nuclear protein in K562 cells and fetal bone marrow which bound to the Ag enhancer [88], and furnished evidence that this protein was Hox-b2 [89]. They could not detect this protein in adult blood cells, suggesting that its expression might be developmentally stage-specific, and that it might be an activator of fetal globin production. However a transcriptional effect of Hox-b2, or any other Hox protein, on gamma globin gene expression has not yet been demonstrated.
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DNA Binding Motifs for Hox Proteins
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Although Hox proteins are thought to be transcription factors, there has been a general failure to define targets and modes of action for these proteins. Most attempts to define a consensus DNA binding sequence have used the homeodomain portion of Hox proteins to identify an ATTA core binding motif, but binding of full-length Hox proteins by themselves is very weak. An exciting development in this area has been the recent discovery that the proto-oncogene Pbx1 acts as a protein partner to facilitate cooperative DNA binding by Hox proteins [90–93]. Pbx1 is a non-Hox class homeodomain protein which was initially described as a fusion protein with a transcriptional activator domain of the E1A protein which occurs in t(1,19) translocations seen in a number of cases of pre-B cell leukemias [11, 94].
Recent studies have shown that Hox proteins cooperatively bind to DNA in the presence of Pbx1 under conditions where the individual proteins bind very weakly or not at all [90, 93]. These experiments have further established that an ATCAATCA octamer serves as a consensus binding site of Hox-pbx heterodimers [91], and that this protein-protein interaction requires a short hexapeptide motif, with a core YPWM sequence, located N terminal to the homeodomain of the Hox protein [92, 95]. Finally, there appears to be a large difference in the relative stabilities of the complexes of various Hox proteins with Pbx1 and DNA with a given DNA target sequence, providing a possible mechanism to explain the differential functions of Hox proteins which share very highly conserved DNA binding domains [95]. The recognition that Hox and Pbx proteins bind DNA as heterodimers should permit more accurate identification of authentic target sites for Hox protein regulation in the genome. These observations do not preclude the possibility that there are other protein partners for Hox peptides yet to be discovered.
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The Biological Function of Hox Proteins in Hematopoietic Cells: Does Blood Have an Axis and Do HOX Cluster Genes Function as Clocks?
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It is somewhat difficult to visualize a role for Hox proteins in blood cells which parallels their proposed role in body plan determination. A major conclusion concerning the role of these genes in development is that they confer positional information along various body axes, thereby regulating morphogenesis. It is not immediately obvious that the hematopoietic system requires positional information or that it has any "axes". However, some analogies can be drawn between embryogenesis and hematopoiesis. Blood cells do go through a carefully coordinated series of morphogenetic movements from the yolk sac to the fetal liver and spleen, and then finally to the bone marrow, processes conceivably controlled in part by HOX genes. It is also noteworthy that adult marrow does have a three-dimensional structure and organization, with the most primitive cells juxtaposed against the endostium, and maturing cells migrating progressively more centrally towards the venous sinuses, into which fully differentiated blood cells are eventually released [96–98]. It is possible that HOX genes coordinate that process by modulating adhesive interactions between blood cells and their neighbors in the marrow.
Another recurring theme in HOX gene expression studies, whether along the A-P axis of the embryo, in the developing limb bud, in retinoic-acid treated ES cells, in activated T cells, or in various subsets of CD34+ marrow cells, is that activation is characterized by a temporally limited wave of gene expression beginning at the 3' end of the HOX locus and proceeding to the 5' end. It is thus conceivable that HOX gene clusters have a general function as biologic clocks, which can be plugged into diverse developmental programs which require a carefully ordered and time-consuming series of sequential transcriptional events.
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Conclusions and Prospectives for Further Study
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There is now ample evidence that HOX genes are expressed in stage- and lineage-specific patterns in normal hematopoietic cells, and that modulating that expression can perturb a number of aspects of blood cell development. The studies to date indicate that the effects of different HOX genes are unique. From a clinical standpoint it is reasonable to speculate that alteration in HOX gene expression could contribute to the pathophysiology of human leukemia, and that patterns of HOX gene activation could be used as molecular markers of specific subtypes of leukemia. Furthermore, the ability to expand stem cells ex vivo by manipulating HOX gene expression could have a major therapeutic role in marrow transplantation and gene therapy. We also predict that mutations of HOX genes may prove to be the bases for a variety of inherited human diseases involving both morphogenetic and hematological abnormalities, such as thrombocytopenia and absent radius syndrome [99].
We anticipate that the next decade will see a number of descriptions of hematopoietic defects in transgenic mice with targeted interruptions of various Hox genes, phenotypes which should further elucidate the physiologic role of these genes. In addition, we expect to see an explosion of information concerning the normal gene targets of Hox proteins, which may include adhesion molecules, growth factors and their receptors, and perhaps even blood cell-specific proteins such as hemoglobin. Given the profound effects of HOX genes on hematopoiesis and embryogenesis, it is reasonable to speculate that target genes in blood cells and embryonic cells include those involved in primary cell processes of development—proliferation, death, motion, adhesion and differentiation—all processes which are more easily studied in a variety of hematopoiesis-systems than in the whole embryo. In fact one might argue that the hematopoietic system may emerge as the most powerful system in which to elucidate general mechanisms of homeobox gene functions.
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Abstract
Introduction
