Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter.
Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.
The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.
Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.
HOX cofactors enhance HOX binding affinities and specificities and increase HOX's unique functional activities. The expression and the regulation of HOX cofactors in human ovaries are unknown.
In this study, the expression of HOX cofactors, PBX1, PBX2, and MEIS1/2, were examined by using RT-PCR, immunofluorescence in cultured immortalized human granulosa (SVOG) cells. The distribution of these HOX cofactors in human ovaries was examined by immunohistochemistry. The effects of growth differentiation factor-9 (GDF-9) and follicle-stimulating hormone (FSH) on PBX2 in SVOG cells were investigated by western blot analysis. Binding activities of HOXA7 and PBX2 to the specific sequences in granulosa cells were determined by electrophoretic mobility shift assay (EMSA).
Results and conclusion
In SVOG cells, PBX1, PBX2 and MEIS1/2 were expressed during cell culture. In normal human ovaries, PBX1 and MEIS1/2 were expressed in granulosa cells at essentially all stages of follicular development. These cofactors were expressed in the nuclei of the granulosa cells from the primordial to the secondary follicles, whereas beyond multilayered follicles was observed in the cytoplasm. The co-expression of PBX1 and MEIS1/2 in granulosa cells in normal human ovaries suggested that MEIS1/2 might control PBX1 sublocalization, as seen in other systems. PBX2 was not expressed or weakly expressed in the primordial follicles. From the primary follicles to the preovulatory follicles, PBX2 expression was inconsistent and the expression was found in the granulosa cell nuclei. The PBX2 expression pattern is similar to HOXA7 expression in ovarian follicular development. Furthermore, FSH down-regulated, GDF-9 did not change PBX2 expression, but co-treatment of the granulosa cells with FSH and GDF-9 up-regulated PBX2 expression. These results implicated a role for PBX2 expression in the steroidogenic activities of granulosa cells in humans. Moreover, PBX2 and HOXA7 bound together to the Pbx sequence, but not to the EMX2 promoter sequence, in SVOG cells. Our findings indicate that HOX cofactors expression in normal human ovary is temporally and spatially specific and regulated by FSH and GDF-9 in granulosa cells. HOX proteins may use different HOX cofactors, depending on DNA sequences that are specific to the granulosa cells.
To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb’s ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation.
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.
c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival of erythroid, myeloid and lymphoid cells, but decreases during the terminal differentiation to mature blood cells. The cellular level of c-Myb is controlled by not only transcriptional regulation but also ubiquitin-dependent proteolysis. We recently reported that mouse c-Myb protein is controlled by ubiquitin-dependent degradation by SCF-Fbw7 E3 ligase via glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of Thr-572 in a Cdc4 phosphodegron (CPD)-dependent manner. However, this critical threonine residue is not conserved in human c-Myb. In this study, we investigated whether GSK3 is involved in the regulatory mechanism for human c-Myb expression.
Human c-Myb was degraded by ubiquitin-dependent degradation via SCF-Fbw7. Human Fbw7 ubiquitylated not only human c-Myb but also mouse c-Myb, whereas mouse Fbw7 ubiquitylated mouse c-Myb but not human c-Myb. Human Fbw7 mutants with mutations of arginine residues important for recognition of the CPD still ubiquitylated human c-Myb. These data strongly suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner. Mutations of the putative GSK3 phosphorylation sites in human c-Myb did not affect the Fbw7-dependent ubiquitylation of human c-Myb. Neither chemical inhibitors nor a siRNA for GSK3β affected the stability of human c-Myb. However, depletion of GSK3β upregulated the transcription of human c-Myb, resulting in transcriptional suppression of γ-globin, one of the c-Myb target genes.
The present observations suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner, whereas mouse Fbw7 ubiquitylates human c-Myb in a CPD-dependent manner. Moreover, GSK3 negatively regulates the transcriptional expression of human c-Myb but does not promote Fbw7-dependent degradation of human c-Myb protein. Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells. We conclude that expression levels of human and mouse c-Myb are regulated via different mechanisms.
Mixed lineage leukemia (MLL) fusion proteins directly activate the expression of key downstream genes such as MEIS1, HOXA9 to drive an aggressive form of human leukemia. However, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion pathway, contribute to the development of MLL leukemia. Here we show that the transcription factor PU.1 is essential for MLL leukemia and is required for the growth of MLL leukemic cells via the promotion of cell-cycle progression and inhibition of apoptosis. Importantly, PU.1 expression is not under the control of MLL fusion proteins. We further identified a PU.1-governed 15-gene signature, which contains key regulators in the MEIS-HOX program (MEIS1, PBX3, FLT3, and c-KIT). PU.1 directly binds to the genomic loci of its target genes in vivo, and is required to maintain active expression of those genes in both normal hematopoietic stem and progenitor cells and in MLL leukemia. Finally, the clinical significance of the identified PU.1 signature was indicated by its ability to predict survival in acute myelogenous leukemia patients. Together, our findings demonstrate that PU.1 contributes to the development of MLL leukemia, partially via crosstalk with the MEIS/HOX pathway.
PU.1; MEIS1; MLL leukemia; transcription regulation
NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both pre-leukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 out of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 out of 27 (30%) acquired a Flt3 mutation. Two CA10 AMLs acquired a Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation.
CALM; AF10; NUP98; HOXD13; Hoxa9; Meis1; Pbx3; acute myeloid leukemia (AML)
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.
c-myb is a frequent target of retroviral insertional mutagenesis in murine leukemia virus-induced myeloid leukemia. Induction of the leukemogenic phenotype is generally associated with inappropriate expression of this transcriptional regulator. Despite intensive investigations, the target genes of c-myb that are specifically involved in development of these myeloid lineage neoplasms are still unknown. In vitro assays have indicated that c-myc may be a target gene of c-Myb; however, regulation of the resident chromosomal gene has not yet been demonstrated. To address this question further, we analyzed the expression of c-myc in a myeloblastic cell line, M1, expressing a conditionally active c-Myb–estrogen receptor fusion protein (MybER). Activation of MybER both prevented the growth arrest induced by interleukin-6 (IL-6) and rapidly restored c-myc expression in nearly terminal differentiated cells that had been exposed to IL-6 for 3 days. Restoration occurred in the presence of a protein synthesis inhibitor but not after a transcriptional block, indicating that c-myc is a direct, transcriptionally regulated target of c-Myb. c-myc is a major target that transduces Myb's proliferative signal, as shown by the ability of a c-Myc–estrogen receptor fusion protein alone to also reverse growth arrest in this system. To investigate the possibility that this regulatory connection contributes to Myb's oncogenicity, we expressed a dominant negative Myb in the myeloid leukemic cell line RI-4-11. In this cell line, c-myb is activated by insertional mutagenesis and cannot be effectively down regulated by cytokine. Myb's ability to regulate c-myc's expression was also demonstrated in these cells, showing a mechanism through which the proto-oncogene c-myb can exert its oncogenic potential in myeloid lineage hematopoietic cells.
HOXA9 plays a critical role in both normal hematopoiesis and leukemogenesis, particularly in the development and maintenance of mixed lineage leukemia (MLL)-rearranged leukemia. Through reverse transcription polymerase chain reaction (RT-PCR) analysis of HOXA9 transcripts in human leukemia and normal bone marrow samples, we identified a truncated isoform of HOXA9, namely HOXA9T, and found that both HOXA9T and canonical HOXA9 were highly expressed in leukemia cell lines bearing MLL rearrangements, relative to human normal bone marrow cells or other subtypes of leukemia cells. A frameshift in HOXA9T in exon I causes a premature stop codon upstream of the PBX binding domain and the homeodomain, which leads to the generation of a non-homeodomain-containing protein. Unlike the canonical HOXA9, HOXA9T alone cannot transform normal bone marrow progenitor cells. Moreover, HOXA9T cannot cooperate with MEIS1 to transform cells, despite the presence of a MEIS1-binding domain. Remarkably, although the truncated isoforms of many proteins function as dominant-negative competitors or inhibitors of their full-length counterparts, this is not the case for HOXA9T; instead, HOXA9T synergized with HOXA9 in transforming mouse normal bone marrow progenitor cells through promoting self-renewal and proliferation of the cells. Collectively, our data indicate that both truncated and full-length forms of HOXA9 are highly expressed in human MLL-rearranged leukemia, and the truncated isoform of HOXA9 might also play an oncogenic role by cooperating with canonical HOXA9 in cell transformation and leukemogenesis.
HOXA9; HOXA9T; isoforms; leukemia
Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.
The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.
Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblast-related genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis.
Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.
•Meis provides a ground state that is common to all the branchial arches•Hoxa2 recognizes Meis prebound sites in the second arch that contain Hox motifs•Hoxa2 enhances Meis binding, which coincides with active enhancers, at these sites•Hoxa2 modulates the ground-state binding of Meis to instruct second arch identity
Hox transcription factors instruct specific morphologies in the branchial arches, a series of segments in the developing vertebrate head. Amin et al. found that Hoxa2 operates as a tissue-specific cofactor, enhancing binding of the homeodomain transcription factor Meis to specific sites that provide the second arch with its anatomical identity.
The interaction of Prep1 and Pbx homeodomain transcription factors regulates their activity, nuclear localization, and likely, function in development. To understand the in vivo role of Prep1, we have analyzed an embryonic lethal hypomorphic mutant mouse (Prep1i/i). Prep1i/i embryos die at embryonic day 17.5 (E17.5) to birth with an overall organ hypoplasia, severe anemia, impaired angiogenesis, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. At E14.5, Prep1 is present in fetal liver (FL) cMyb-positive cells, whose deficiency causes a marked hematopoietic phenotype. Prep1 is also localized to FL endothelial progenitors, consistent with the observed angiogenic phenotype. Likewise, at the same gestational day, Prep1 is present in the eye cells that bear Pax6, implicated in eye development. The levels of cMyb and Pax6 in FL and in the retina, respectively, are significantly decreased in Prep1i/i embryos, consistent with the hematopoietic and eye phenotypes. Concomitantly, Prep1 deficiency results in the overall decrease of protein levels of its related family member Meis1 and its partners Pbx1 and Pbx2. As both Prep1 and Meis interact with Pbx, the overall Prep1/Meis-Pbx DNA-binding activity is strongly reduced in whole Prep1i/i embryos and their organs. Our data indicate that Prep1 is an essential gene that acts upstream of and within a Pbx-Meis network that regulates multiple aspects of embryonic development.
E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.
Complex genetic and biochemical interactions between HOX proteins and members of the TALE (i.e., PBX and MEIS) family have been identified in embryonic development, and some of these interactions also appear to be important for leukemic transformation. We have previously shown that HOXA9 collaborates with MEIS1 in the induction of acute myeloid leukemia (AML). In this report, we demonstrate that HOXB3, which is highly divergent from HOXA9, also genetically interacts with MEIS1, but not with PBX1, in generating AML. In addition, we show that the HOXA9 and HOXB3 genes play key roles in establishing all the main characteristics of the leukemias, while MEIS1 functions only to accelerate the onset of the leukemic transformation. Contrasting the reported functional similarities between PREP1 and MEIS1, such as PBX nuclear retention, we also show that PREP1 overexpression is incapable of accelerating the HOXA9-induced AML, suggesting that MEIS1 function in transformation must entail more than PBX nuclear localization. Collectively, these data demonstrate that MEIS1 is a common leukemic collaborator with two structurally and functionally divergent HOX genes and that, in this collaboration, the HOX gene defines the identity of the leukemia.
Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.
Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice.
E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.
The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.
transcription factor; oncogene; chronic myeloid leukemia
MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.