The Oncoprotein E2A-Pbx1a Collaborates with Hoxa9 To Acutely Transform Primary Bone Marrow Cells

A recurrent translocation between chromosome 1 (Pbx1) and 19 (E2A) leading to the expression of the E2A-Pbx1 fusion oncoprotein occurs in approximately 5 to 10% of acute leukemias in humans. It has been proposed that some of the oncogenic potential of E2A-Pbx1 could be mediated through heterocomplex formation with Hox proteins, which are also involved in human and mouse leukemias. To directly test this possibility, mouse bone marrow cells were engineered by retroviral gene transfer to overexpress E2A-Pbx1a together with Hoxa9. The results obtained demonstrated a strong synergistic interaction between E2A-Pbx1a and Hoxa9 in inducing growth factor-independent proliferation of transduced bone marrow cells in vitro and leukemic growth in vivo in only 39 +/- 2 days. The leukemic blasts which coexpress E2A-Pbx1a and Hoxa9 showed little differentiation and produced cytokines such as interleukin-3, granulocyte colony-stimulating factor, and Steel. Together, these studies demonstrate that the Hoxa9 and E2A-Pbx1a gene products collaborate to produce a highly aggressive acute leukemic disease.     

Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Here we demonstrate that Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. Our results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis.     

Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.     

NUP98-HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid leukemias in mice

Here we describe hemopoietic chimeras serving as a mouse model for NUP98-HOXA9-induced leukemia, which reproduced several of the phenotypes observed in human disease. Mice transplanted with bone marrow cells expressing NUP98-HOXA9 through retroviral transduction acquire a myeloproliferative disease (MPD) and eventually succumb to acute myeloid leukemia (AML). The NUP98 portion of the fusion protein was shown to be responsible for transforming a clinically silent pre-leukemic phase observed for Hoxa9 into a chronic, stem cell-derived MPD. The co-expression of NUP98-HOXA9 and Meis1 accelerated the transformation of MPD to AML, identifying a genetic interaction previously observed for Hoxa9 and Meis1. Our findings demonstrate the presence of overlapping yet distinct molecular mechanisms for MPD versus AML, illustrating the complexity of leukemic transformation.     

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.     

HOXA9 Modulates Its Oncogenic Partner Meis1 To Influence Normal Hematopoiesis

While investigating the mechanism of action of the HOXA9 protein, we serendipitously identified Meis1 as a HOXA9 regulatory target. Since HOXA9 and MEIS1 play key developmental roles, are cooperating DNA binding proteins and leukemic oncoproteins, and are important for normal hematopoiesis, the regulation of Meis1 by its partner protein is of interest. Loss of Hoxa9 caused downregulation of the Meis1 mRNA and protein, while forced HOXA9 expression upregulated Meis1. Hoxa9 and Meis1 expression was correlated in hematopoietic progenitors and acute leukemias. Meis1^+/− Hoxa9^−/− deficient mice, generated to test HOXA9 regulation of endogenous Meis1, were small and had reduced bone marrow Meis1 mRNA and significant defects in fluorescence-activated cell sorting-enumerated monocytes, mature and pre/pro-B cells, and functional B-cell progenitors. These data indicate that HOXA9 modulates Meis1 during normal murine hematopoiesis. Chromatin immunoprecipitation analysis did not reveal direct binding of HOXA9 to Meis1 promoter/enhancer regions. However, Creb1 and Pknox1, whose protein products have previously been reported to induce Meis1, were shown to be direct targets of HOXA9. Loss of Hoxa9 resulted in a decrease in Creb1 and Pknox1 mRNA, and forced expression of CREB1 in Hoxa9^−/− bone marrow cells increased Meis1 mRNA almost as well as HOXA9, suggesting that CREB1 may mediate HOXA9 modulation of Meis1 expression.While the Hox homeobox genes are widely recognized as important developmental genes (26), we and others have shown that several Hox genes, and Hoxa9 in particular, are important for both normal hematopoiesis (27, 28) and leukemic transformation (25, 29). While the Hoxa9 gene plays a role in embryonic development, much of the research on this gene has focused on its role as an oncogene that is often upregulated in acute myeloid leukemias (12, 29). In an analysis of 6,817 genes, Hoxa9 was the most highly positively correlated with treatment failure in acute myeloid leukemia patients (18). Meis1 is a member of the TALE family of non-Hox homeobox genes, which was initially identified as a frequent viral integration site in myeloid leukemias arising in BXH2 mice (32). The Hoxa9 gene is also upregulated in many of the leukemias arising in the BXH2 animals (33). Forced expression of HOXA9 in murine bone marrow (BM) cells in culture results in immortalization of myeloid progenitor cells (4, 15), while transplantation of HOXA9-infected BM cells results in the eventual induction of acute myelogenous leukemia (25). In contrast, transplantation of BM cells infected with HOXA9 plus MEIS1 results in rapid development of disease (25). Both HOXA9 and MEIS1 are expressed following forced expression of the MLL oncogene (47) or in patients with MLL gene rearrangements (22).Hoxa9 is expressed in numerous tissues during development, including rib (8), limb (17), motor neuron progenitors (10), reproductive tract (9), and mammary gland (7). Hoxa9 is also expressed in normal adult BM (24, 43), and loss of Hoxa9 leads to multiple relatively mild defects in normal hematopoiesis (23, 27, 28). Retroviral expression studies have also shown that HOXA9 and MEIS1 are important for myeloid blood cell differentiation (3, 4). Despite the broad expression of Hoxa9 and other Hox genes, relatively little is known about how the HOX proteins function. An important advance was the discovery that many HOX proteins gain DNA binding specificity by forming complexes with the PBX (6, 31), MEIS1 (41), and PREP1 (2) proteins. Although HOXA9 is capable of binding DNA alone (42), it forms cooperative DNA binding complexes with MEIS1 alone (41) and in a triple complex with PBX proteins (40, 44). Despite these apparent advances, relatively few downstream targets for HOX proteins, and HOXA9 in particular (11), have been confirmed.During ongoing studies of the mechanism of action of the HOXA9 protein, we discovered that HOXA9 appeared to upregulate the Meis1 mRNA and protein. Given the numerous biological connections between HOXA9 and MEIS1, we embarked on studies to explore this pathway. Forced expression of HOXA9 in BM cells upregulated the Meis1 mRNA and protein, while loss of Hoxa9 resulted in a reduction in the Meis1 mRNA and protein. In addition, in a biological model to assess Hoxa9 modulation of Meis1, compound mutant animals that were homozygous null at the Hoxa9 locus and heterozygous at the Meis1 locus showed a significant loss of murine BM monocytes, mature B cells, and pre/pro-B-cell progenitors and an increase in orthochromatophilic erythroblasts in postnatal-day-15 mice compared to results for all controls, suggesting that HOXA9 regulates Meis1 during normal hematopoiesis. Chromatin immunoprecipitation (ChIP) analysis did not show direct binding of HOXA9 to distal or proximal Meis1 genomic regions. However, these studies, together with PCR analysis, showed that HOXA9 binds to and upregulates two genes, Creb1 and Pknox1 (the protein product is subsequently referred to as PREP1), whose protein products have previously been reported to upregulate Meis1 expression (13, 14). Addition of CREB1 to Hoxa9^−/− bone marrow cells increased Meis1 mRNA nearly as effectively as HOXA9. Taken together, our data show that HOXA9 indirectly modulates its DNA binding and oncogenic partner MEIS1 and that the DNA-binding property of HOXA9 is required for this process. We further show that Hoxa9 modulation of Mes1 is biologically important during normal hematopoiesis and that CREB1 may mediate the regulation of Meis1 by HOXA9.     

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.     

Altered neuronal lineages in the facial ganglia of Hoxa2 mutant mice

Neurons of cranial sensory ganglia are derived from the neural crest and ectodermal placodes, but the mechanisms that control the relative contributions of each are not understood. Crest cells of the second branchial arch generate few facial ganglion neurons and no vestibuloacoustic ganglion neurons, but crest cells in other branchial arches generate many sensory neurons. Here we report that the facial ganglia of Hoxa2 mutant mice contain a large population of crest-derived neurons, suggesting that Hoxa2 normally represses the neurogenic potential of second arch crest cells. This may represent an anterior transformation of second arch neural crest cells toward a fate resembling that of first arch neural crest cells, which normally do not express Hoxa2 or any other Hox gene. We additionally found that overexpressing Hoxa2 in cultures of P19 embryonal carcinoma cells reduced the frequency of spontaneous neuronal differentiation, but only in the presence of cotransfected Pbx and Meis Hox cofactors. Finally, expression of Hoxa2 and the cofactors in chick neural crest cells populating the trigeminal ganglion also reduced the frequency of neurogenesis in the intact embryo. These data suggest an unanticipated role for Hox genes in controlling the neurogenic potential of at least some cranial neural crest cells.