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1Department of Internal Medicine, Division of Hematology and Oncology, 2Department of Veterinary Biosciences, 3Integrated Biomedical Science Graduate Program, 4Department of Molecular Virology, Immunology and Medical Genetics, 5Comprehensive Cancer Center, 6Department of Molecular Genetics, and 7Division of Biometrics, The Ohio State University, Columbus, Ohio, USA.
Abstract
We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%–7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. MllPTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. MllPTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.
Introduction
Mixed-lineage leukemia (MLL, also known as ALL-1, HRX, or HTRX1) is the human homolog of Drosophila Trx and is a maintenance factor for the homeobox (Hox) group of proteins, which play an important role in specifying cell fate during development and hematopoiesis (1). Approximately 4% of patients with de novo acute myeloid leukemia (AML) have balanced translocations or insertions that result in fusion of MLL at chromosome 11q23 with 1 of over 40 functionally divergent genes. Although a central mechanism responsible for malignant transformation as a result of these chromosomal translocations involving 11q23 is lacking, the N terminus of MLL, which contains the AT-hook DNA-binding motif and a region homologous to DNA methyltransferase, is always retained in the fusion protein while the C terminus, which contains the activation and SET domains, is always replaced by the fusion partner (2).
We discovered a rearrangement of MLL in AML patients whereby MLL is not fused with a partner gene but rather is consistently elongated with an in-frame partial tandem duplication (PTD) of exons 11-5 or 12-5 (former exon designations were 6-2 and 8-2) (3). In contrast with the myriad of MLL fusions in AML that result from chromosomal translocations, AML blasts with the MLL PTD retain the protein’s C terminus, which contains the activation and SET domains and partially duplicates a region within the N terminus containing the AT-hook DNA-binding and repression domains (2). Approximately 4%–7% of AML patients with normal cytogenetics harbor the MLL PTD and carry an especially poor prognosis (4, 5).
HoxA genes are frequently overexpressed in leukemia (6). The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemogenesis is currently unknown, but given MLL’s role as a maintenance factor for Hox proteins, 1 mechanism could involve deregulation of Hox gene expression. In in vitro model systems, WT MLL regulates certain HOX gene expression via epigenetic modification at cis-regulatory regions, i.e., histone H3/H4 acetylation and H3 lysine 4 (Lys4) methylation, the latter of which requires MLL’s C terminus SET domain (7, 8). When the MLL-AF9 fusion gene (the result of one of the most common MLL rearrangements) is transduced into Mll–/– cells, Hoxa9 is overexpressed by an unknown mechanism that does not require binding of the MLL-AF9 fusion directly to the Hoxa9 promoter (8). However, in another in vitro study, transduction of the MLL–eleven nineteen leukemia and the MLL–FK506-binding protein fusions into MllWT/WT cells increased Hoxa9 expression via H3 acetylation but without increased H3 Lys4 methylation (9). Likewise, when the Mll-AF9 fusion was knocked in as an endogenous allele, mice showed Hoxa9 overexpression and developed AML, but no epigenetic or other alteration could account for this overexpression (10, 11). This suggests distinct mechanisms for induction of HOX genes by WT MLL and fusions of MLL resulting from balanced chromosomal translocations.
Taspase 1 cleaves the approximately 430-kDa MLL protein into 2 smaller components, and this cleavage has been shown to be essential for normal MLL function and regulation. Absence or downregulation of taspase 1 leads to absence or decreases in transcription of normal downstream targets of MLL, such as the Hox genes (12). In virtually all cases involving reciprocal MLL translocations, the consensus sequences for taspase 1 are lost and replaced by the fusion partner, resulting in one of the only known common features among all the reciprocal translocations involving MLL. However, the MLL PTD differs from translocations in that the consensus sequences for taspase 1 are retained and the MLL PTD protein is cleaved (13). How these MLL mutations function with or without taspase 1 cleavage is currently unsolved but highlights another important difference between MLL fusions and the MLL PTD.
In this study, we created a mouse expressing the Mll PTD off of the endogenous Mll promoter and examined the molecular basis for its phenotype. We show that this mouse has overexpression of certain HoxA genes, axial skeletal defects, and aberrant hematopoiesis, all compared with age- and sex-matched littermate controls. We also show that the Mll PTD is associated with increased histone H3/H4 acetylation and methylation of H3 Lys4 at cis-regulatory HoxA sequences, providing what we believe to be the first in vivo evidence for a mechanism by which an MLL gene rearrangement can directly alter HoxA gene expression.
Results
MllPTD/WT mice are viable and express the fusion transcript. To study the in vivo consequences of the Mll PTD and to more closely model the monoallelic involvement seen in human AML with the MLL PTD (14), we employed homologous recombination in ES cells to introduce a genomic fragment containing exons 5 through 11 into intron 4 of Mll (Figure 1). Using 2 MllPTD/WT(+) ES clones, we obtained 11 mice with germline transmission of the Mll PTD and backcrossed these mice with C57BL/6J mice to obtain MllPTD/WT mice on a pure (congenic) C57BL/6J background. Southern blotting (Figure 2A) and DNA PCR (not shown) verified the presence of the MllPTD/WT genotype. Southern blotting on 1-day-old pups from 5 matings also confirmed that MllPTD/PTD homozygous mice are embryonic lethal (Table 1). Sequencing of the RT-PCR product from MllPTD/WT mice demonstrated a correctly spliced, in-frame fusion transcript confirming the precise targeting of exons 5 through 11 within intron 4 of the Mll locus (Figure 2B).
Figure 1
Generation of MllPTD/WT mice. The backbone of the targeting vector is ploxPneo1 and contains a genomic clone spanning Mll intron 4 through intron 11. Linearization of the vector at a unique SfiI site within intron 4 allows for recombination at the germline intron 4 of Mll. A plasmid vector expressing Cre recombinase from a CMV promoter (pCre) was used to excise the neo cassette.
Figure 2
Germline transmission and verification of correct targeting in mice heterozygous for the Mll PTD. (A) Southern blot analysis using high molecular-weight DNA from spleens, digested with NdeI and hybridized to a probe that spans intron 4 and exon 5 of Mll (i4e5 in Figure 1). This generates a single WT band at 18 kb in MllWT/WT mice, 2 bands representing 1 WT allele at 18 kb, and a band at 40 kb representing the rearranged allele in MllPTD/WT mice. (B) The Mll PTD fusion transcript was amplified using an upstream primer from Mll exon 11 and a downstream primer from Mll exon 6 amplifying a single 335-bp unique fusion transcript in the MllPTD/WT mouse (+) and is absent in the sample from the MllWT/WT mouse (–). Sequencing of these PCR products from the MllPTD/WT mice verified the presence of the exon 11–exon 5 fusion site. The 1-kb+ ladder was used to determine amplification size.
Table 1
Assessment of Mendelian ratios for the Mll PTD allele
Absolute quantification of WT and PTD transcripts in MllPTD/WT mice using real-time RT-PCR. Real-time primers and probes were designed and optimized, amplifying the unique 11-5 Mll PTD fusion present only in MllPTD/WT mice as well as the 14-15 Mll exon-exon junction located outside the duplicated region and common to both the Mll PTD allele and the Mll WT allele. Using these probes in an absolute quantification assay (13), it was determined that the Mll PTD and Mll WT alleles were both expressed at low, near equivalent levels in the hematopoietic tissues of the MllPTD/WT mice (Figure 3 and Table 2). Protein confirmation of this equal yet low level of gene expression by Western blot was not technically possible.
Figure 3
Mll WT and Mll PTD transcripts. Schematic demonstrating the real-time RT-PCR strategy for detection and absolute quantification of the Mll WT and Mll PTD transcripts in bone marrow and spleen of Mll PTD mice. The predicted Mll PTD and Mll WT allele–derived transcripts are shown, with the tandemly duplicated exons 5–11 present in the PTD transcript denoted with light gray boxes. Shown below the transcripts are sites for PCR primers and fluorogenic probes designed to amplify either the unique exon 11–exon 5 fusion (open rectangle) or exons 14–15, whose junction is common to the Mll WT and Mll PTD transcripts (filled rectangle).
Table 2
Absolute quantitation of Mll PTD and Mll WT transcripts
Phenotypic abnormalities of MllPTD/WT mice. Examination of MllPTD/WT mice revealed skeletal abnormalities compared with MllWT/WT age- and sex-matched littermate controls. Radiographic examination of MllPTD/WT mice less than 1 year of age identified axial skeleton malformations, including a rudimentary or missing thirteenth rib, indicative of a T13L1 transformation (70% penetrance, n = 13), and an S1 vertebral duplication, indicative of an S2S1 transformation (63%, n = 11) (Figure 4, B and C).
Figure 4
Skeletal analysis of MllPTD/WT mice at 20 weeks of age. (A) Alizarin red staining of MllPTD/WT (left) and MllWT/WT (right) mice shows a rudimentary/missing thirteenth rib, indicative of a T13 L1 transformation (penetrance of 70% in MllPTD/WT mice, n = 13). MicroCT images of the sacral spine in a (B) MllPTD/WT mouse and a (C) MllWT/WT mouse in 3D. These images are viewed from the dorsal side and illustrate the duplication of the S1 vertebral body, indicative of an S2S1 transformation in the MllPTD/WT mice (penetrance of 63%, n = 11).
Given the WT Mll’s role in maintaining Hox expression during development and the known association between HoxA expression, anteroposterior patterning, and posterior axis elongation, we examined Hoxa9 expression during embryogenesis. In situ hybridization performed on E12.5 MllPTD/WT embryos demonstrated a shifted expression boundary of Hoxa9 within the paraxial mesoderm compared with MllWT/WT littermate controls (Figure 5).
Figure 5
In situ hybridizations for Hoxa9 on E12.5 embryos. (A) MllWT/WT and (B) MllPTD/WT embryos hybridized with a digoxygenin-labeled probe for Hoxa9. Results demonstrate a notable ventral shift of the Hoxa9 expression boundary within the paraxial mesoderm of the MllPTD/WT embryo compared with the MllWT/WT littermate control (see inset for each figure). This figure represents 1 of 3 comparable hybridizations.
Altered hematopoiesis in MllPTD/WT mice is associated with aberrant HoxA expression. Lineage-specific CFU assays were performed for erythroid (measured by erythropoietic burst formation [BFU-E]), myeloid, (measured by GM-CFU), and mixed (measured by granulocyte, erythroid, macrophage, megakaryocyte–CFU [GEMM-CFU]) progenitor populations, using both MllPTD/WT and MllWT/WT splenocytes. The CFU number for each progenitor type was significantly increased in MllPTD/WT mice compared with age- and sex-matched MllWT/WT littermate controls (Figure 6A). A 5- to 20-fold increase in MllPTD/WT spleen cells expressing the erythroid marker Ter119 was also noted, consistent with the observed increase in erythroid progenitor populations as measured by BFU-E (Figure 6, B and C). However, extensive phenotypic analyses performed as previously described (15) on blood, spleen, and BM did not reveal any other differences in MllPTD/WT mice compared with age- and sex-matched MllWT/WT littermate controls (see representative flow cytometric analyses, Supplemental Figure 1; available online with this article; doi:10.1172/JCI25546DS1). Primary CFUs were then harvested and used in 2 parallel secondary assays. In replating CFU assays, the total number of colonies per plate was not significantly different between MllPTD/WT and MllWT/WT splenocytes, yet the size of the MllPTD/WT colonies was markedly increased over both MllWT/WT colonies and those derived from MllAf9/WT mice (Figure 6, D–F). Secondary colonies from MllPTD/WT mice were able to form tertiary and quaternary colonies while those from MllWT/WT and MllAf9/WT mice were not (data not shown). The increase in size per MllPTD/WT colony suggested an increase in progenitor cell proliferation compared with MllWT/WT progenitor cells. Nonetheless, absolute in vivo cell counts in bone marrow, spleen, and blood were not significantly different between MllPTD/WT and MllWT/WT mice, suggesting that enhanced proliferation in the MllPTD/WT progenitor cells was accompanied by increased cell turnover. To verify this, primary CFU colonies were harvested and maintained in liquid cultures supplemented with SCF, IL-3, and IL-6 for an additional 18 days, followed by cell counts, measurement of BrdU uptake for quantification of proliferation, and assessment of apoptosis by annexin V and propidium iodide staining. The MllPTD/WT hematopoietic progenitor cells did have a 2-fold greater fraction of cells incorporating BrdU compared with MllWT/WT progenitor cells (Figure 6, G–H) but also showed a substantial amount (50%) of apoptosis (Figure 6I) such that absolute cell counts over this period of time were only modestly increased from the initiation of culture (Table 3). This near maintenance of homeostasis could also explain the normal cell counts observed in bone marrow, spleen, and blood of MllPTD/WT mice in vivo. The lack of viable MllWT/WT cells at day 18 in repeated assays (Table 3) precluded our ability to obtain statistically meaningful comparative data in repeated cultures. In contrast, MllPTD/WT progenitor cells were sustained in liquid culture for more than 4 months.
Figure 6
Evaluation of progenitor populations in MllPTD/WT splenocytes compared with MllWT/WT sex-matched littermate controls. (A) Results from CFU assays to assess progenitors of erythroid (BFU-E), granulocytic, erythroid, monocytic, megakaryocytic (GEMM-CFU), and granulocyte, macrophage (GM-CFU) lineages show significantly increased CFUs derived from MllPTD/WT versus MllWT/WT splenocytes. *P = 0.03; **P = 0.0025. Error bars indicate SD. Splenocytes from (B) MllWT/WT and (C) MllPTD/WT mice (n = 8 mice per genotype) show increased surface density expression of the erythroid marker Ter119 in MllPTD/WT mice. Ter PE, PE fluorochrome–conjugated Ter119. Secondary splenic colonies from (D) MllWT/WT, (E) MllAf9/WT, and (F) MllPTD/WT mice (n = 6 mice per genotype, plated in duplicate). The numbers of secondary colonies per plate (~230) were similar between genotypes. The number of cells per colony was dramatically increased in MllPTD/WT mice compared with MllWT/WT or MllAf9/WT mice. (G–I) Primary CFU progenitor cells harvested after 14 days and maintained in liquid cultures for 18 days, supplemented with SCF, IL-3, and IL-6, and in the presence of BrdU for the last 4 days. An anti-BrdU antibody gated on viable cells demonstrates a smaller fraction of MllWT/WT cells proliferating (G) compared with MllPTD/WT cells (H). Assessment of programmed cell death (I, upper right and lower right quadrants) revealed a sizeable fraction (~50%) of MllPTD/WT progenitors undergoing apoptosis during expansion. While a significant fraction of MllPTD/WT cells were proliferating, approximately 50% were undergoing apoptosis. In 2 additional experiments, the MllWT/WT cells had already died by this time point in culture (see Table 3), precluding a statistical comparison of these results in G–I.
Table 3
Splenic progenitor cells grown in liquid culture
Increased expression of HoxA genes in MllPTD/WT mice. To establish a possible cause for the aberrant increases in both the number of primary colonies and increased proliferative capacity of replated progenitors from MllPTD/WT mice, we quantified expression of Hoxa7, Hoxa9, and Hoxa10 within hematopoietic tissues. We found consistent and significant overexpression of each HoxA gene in BM, spleen, and blood when compared with their expression in matched MllWT/WT littermate controls (Figure 7, A–C). Expression of Hoxa1, a HoxA family member that is not regulated by Mll, was unaltered in any of the hematopoietic tissues examined while Hoxc8, a HoxC family member that has been shown to be positively regulated by WT MLL (8), was also not upregulated in any of the tissues examined (not shown).
Figure 7
HoxA gene expression levels. (A–C) Quantification of Hoxa7, Hoxa9, and Hoxa10 expression in hematopoietic tissues of MllWT/WT and MllPTD/WT mice. Cells were isolated from (A) BM, (B) spleen, and (C) blood from both MllWT/WT and MllPTD/WT mice (n = 10 mice per genotype). RNA and then cDNA were prepared and quantified by real-time RT-PCR. MllPTD/WT mice showed increased expression of each of these HoxA genes in all 3 tissues compared with sex-matched littermate MllWT/WT mice. (D) Equal numbers of each sorted BM population expressing Gr-1, CD11b, CD3, B220, or Ter119 from MllPTD/WT mice and MllWT/WT mice were processed for RNA and then cDNA. Absolute quantification of Hoxa9 expression was then determined by real-time RT-PCR and demonstrated that MllPTD/WT cells have increased Hoxa9 expression within each population compared with the equivalent number of MllWT/WT cells, indicating that Hoxa9 transcript levels are overexpressed on a per cell basis (values shown represent mean of n = 3 mice per genotype ± SD; P < 0.03).
The lack of significant differences in cellular populations of blood, spleen, and BM between MllPTD/WT and MllWT/WT mice as noted above suggested that the increased HoxA gene expression was due to an increase in the HoxA expression on a per cell basis. To confirm this, we sorted equal numbers of cells from BM of MllPTD/WT and MllWT/WT mice expressing different lineage markers: B220, CD3, CD11b, Gr-1, and Ter119. RNA was isolated from an equivalent number of cells and reverse transcribed into cDNA. Using real-time RT-PCR, Hoxa9 was shown to be overexpressed in each of the MllPTD/WT cell subsets when compared with the identical MllWT/WT subset, consistent with an increase in HoxA gene expression on a per cell basis (Figure 7D).
Epigenetic alterations at HoxA gene promoters in MllPTD/WT mice. As noted earlier, HoxA gene overexpression has been noted in both in vitro and in vivo models of the MLL-AF9 fusion, but an in vivo mechanism to explain this upregulation in this or any MLL rearrangement associated with leukemia has yet to be provided (11). Two groups have shown that WT MLL has intrinsic histone H3 Lys4 methyltransferase activity within its C terminus SET domain (7, 8), which is retained in the MLL PTD. To test whether epigenetic modifications within HoxA promoters were responsible for HoxA overexpression in MllPTD/WT hematopoietic tissues, chromatin immunoprecipitation (ChIP) was performed within promoter sequences known to be important for maintaining HoxA transcription and spleen and BM were compared with similar tissues in MllWT/WT mice. MllPTD/WT mice displayed an increase in histone H3 and H4 acetylation and H3 Lys4 methylation with a corresponding decrease in H3 Lys9 methylation within the Hoxa9 (Figure 8, A and B) and Hoxa7 (not shown) promoters. These epigenetic modifications associated with gene transcription establish a mechanistic link between the MllPTD/WT genotype and the associated overexpression of Mll targets Hoxa7 and Hoxa9. No such epigenetic changes were noted within the pertinent promoter region of Hoxa10.
Figure 8
Assessment of Mll target gene Hoxa9 acetylation and methylation of histones assessed by ChIP using (A) bone marrow cells and (B) splenocytes derived from MllPTD/WT and MllWT/WT mice. Results show an increase at the Hoxa9 promoter in histone H3/H4 acetylation as well as histone H3 Lys4 methylation and a corresponding decrease in histone H3 Lys9 methylation in MllPTD/WT bone marrow and spleen compared with the same MllWT/WT tissues. Results are representative of 3 independent experiments. Similar results were obtained at the Hoxa7 promoter but were not seen at the Hoxa10 promoter (not shown). (C) BM cells and (D) splenocytes derived from MllPTD/WT, MllWT/–, and MllWT/WT mice. Absolute quantification by SYBR green real-time PCR shows a significant increase at the Hoxa9 promoter in histone H3/H4 acetylation as well as histone H3 Lys4 methylation in MllPTD/WT BM and spleen compared with the same MllWT/– and MllWT/WT tissues (n = 3 mice per genotype; P < 0.05). There was no significant difference between MllWT/– and MllWT/WT tissues.
We further assessed whether the increased histone modifications associated with gene activation were due to a gain of function of the Mll PTD and not the result of losing a WT Mll allele. H3/H4 acetylation and H3 Lys4 methylation at the Hoxa7 and Hoxa9 promoters were therefore measured using ChIP in combination with Sybr green real-time PCR in MllPTD/WT and MllWT/– BM and spleen and compared with similar measurements in MllWT/WT BM and spleen. Results show no significant difference in the histone modifications at these promoters when comparing MllWT/– and MllWT/WT tissues. In contrast, MllPTD/WT tissues showed significantly increased H3/H4 acetylation and H3 Lys4 methylation at the Hoxa7 and Hoxa9 promoters when compared with these other 2 genotypes (Figure 8, C and D, and Table 4). These data indicate that the histone modifications associated with gene activation in the MllPTD/WT cells result from gain-of-function mutation involving the PTD and not simply the loss of a WT Mll allele. It should also be noted that HoxA gene expression in MllPTD/WT E17.5 fetal liver cells showed a 5- to 10-fold increase in Hoxa7, Hoxa9, and Hoxa10 gene expression compared with either MllWT/WT or MllWT/– fetal liver cells (data not shown). Finally, preliminary experiments comparing fetal liver cell HoxA gene expression obtained from MllPTD/WT, MllWT/–, and MllPTD/– E17.5 embryos strongly support the conclusion that the Mll PTD confers a gain-of-function mutation (A. Dorrance and M. Caligiuri, unpublished observations).
Table 4
Assessment of MllPTD/WT and MllWT/– phenotypes compared with MllWT/WT genotype
Absence of leukemic transformation in MllPTD/WT mice highlights important requirement for second hit in the progression of leukemia. Despite the aberrant hematopoiesis, the mice had not developed leukemia over 2 years of observation. Hoxa9 cooperates with overexpressed Meis1 in the induction of murine AML (16–18). We therefore quantified Meis1 transcript in affected hematopoietic tissues but found no evidence of overexpression (not shown). Likewise, we have noted that AML blasts from patients harboring the MLL PTD consistently lack expression of the WT MLL allele (13); however, hematopoietic tissues of MllPTD/WT mice showed near equivalent levels of WT and PTD transcript (Figure 2C). Thus, it is possible that these and/or other additional molecular alterations are required for malignant transformation and can explain the aberrant hematopoiesis in the absence of leukemic transformation in MllPTD/WT mice.
Discussion
In the current report, we describe a mouse engineered with Mll PTD expression driven by its endogenous promoter. The MllPTD/PTD genotype appears embryonic lethal, and characterization of these embryos is ongoing. MllPTD/WT mice are viable, are able to reproduce, and are without serious illness after 2 years of observation. However, MllPTD/WT mice have a high penetrance of skeletal abnormalities that consist of a missing or rudimentary thirteenth rib and a duplication of the S1 vertebral body.
Hematopoietic abnormalities consist of significantly increased BFU-E, GM-CFU, and GEMM-CFU progenitors in cultures from MllPTD/WT splenocytes when compared with age- and sex-matched MllWT/WT littermate controls. Alterations in members of the homeobox or Hox family of genes could reasonably explain these seemingly diverse phenotypic findings in the Mll PTD mice since certain Hox members are critical in axioskeletal and hematopoietic development and maintenance of Hox expression is positively regulated by WT Mll (1). The current report documents the increased expression of selected HoxA genes that is coincident with the Mll PTD and, for what we believe is the first time, provides a mechanism by which an Mll defect that is associated with AML causes deregulation of HoxA expression in vivo.
Hox genes display spatiotemporal expression patterns that are colinear with respect to their location on the chromosome (19). Thus, for normal skeletal development to proceed, Hox genes must be expressed in the right cell type at the right time. Expression of these genes at incorrect times or locations leads to disruptions in normal Hox expression boundaries, resulting in altered cell fate and misspecification of segment identities (20–22). To determine whether the malformations of the axial skeletons of our MllPTD/WT mice were associated with alterations in Hox expression boundaries, we performed whole-mount in situ hybridizations on E12.5 embryos and found the expression of Hoxa9 in the MllPTD/WT embryos extended laterally in the somitic mesoderm when compared with MllWT/WT littermate controls. This finding is consistent with other reports that have implicated Hoxa9 as an important gene for lumbosacral patterning (23).
Restricted Hox expression is also vital for normal hematopoiesis, and deregulation in HoxA expression can result in aberrant hematopoiesis and malignant transformation (18, 24–30). Following the observations of progenitor cell abnormalities in MllPTD/WT spleen, we noted significant increases in Hoxa7, Hoxa9, and Hoxa10 transcripts in the spleen, BM, and blood of MllPTD/WT mice when compared with MllWT/WT littermate controls and with MllWT/– mice. However, Hoxa1, a HoxA family member not regulated by Mll, showed no increase in its expression in tissues of the MllPTD/WT mice. Likewise, Hoxc8, a member of the HoxC paralogous group that has been shown to be a direct target of MLL, was not increased within these tissues, indicating the specific effect of the Mll PTD on these HoxA genes. Further, as these HoxA family members are positively regulated by WT Mll, their consistent and significant overexpression in the tissues of MllPTD/WT mice also suggests that the Mll PTD was operating in a gain-of-function fashion.
WT MLL has been shown to directly and indirectly modify histones at the promoters of certain HOX genes, thereby maintaining gene transcription (7, 8). Histone modifications are associated with gene activation and repression (31–33). Histone acetylation is the predominant modification associated with gene activation as well as histone H3 Lys4 methylation, while histone H3 Lys9 methylation corresponds with gene repression. Enzymes known as histone acetyltransferases, such as cAMP response element–binding protein–binding protein (CBP), are responsible for histone acetylation and associate with MLL in a complex via its C terminal transactivation domain, which is uniquely preserved in the MLL PTD (34). There are genetic data showing evolutionarily conserved association of the MLL homolog Trx with Drosophila CBP in the fly, thus demonstrating the importance of this protein interaction (35). Other complexes found within the MLL supercomplex function as either activators or repressors of transcription, some via acetylation (7, 8). Examination of the histone acetylation states at Hoxa7 and Hoxa9 promoters in our study showed overacetylation of histone H3/H4 in spleen and BM of MllPTD/WT mice, explaining at least 1 mechanism by which the Mll PTD is mediating HoxA overexpression. The specific complexes associating with the Mll PTD that are responsible for this observation and the direct or indirect mechanism by which this is accomplished have yet to be elucidated.
Like the transactivation domain, the SET domain is also retained in the C terminus of the MLL PTD, again in contrast to all other MLL gene fusions found in AML. The SET domain of MLL has histone H3 Lys4 methyltransferase activity essential for maintaining Hox expression (7, 8). We hypothesized that histone H3 Lys4 methylation might also contribute to upregulation of these HoxA genes. Indeed, ChIP experiments revealed a correlation between overexpression of both Hoxa7 and Hoxa9 transcript with increases of histone H3 Lys4 methylation at these promoters in MllPTD/WT tissues. These data and the absence of such an increase in the same tissues of MllWT/– mice support the notion that the Mll PTD has a direct gain-of-function role in transcriptional activation of these target genes.
In contrast, the retroviral infection of Mll–/– murine embryonic fibroblasts with the MLL-AF9 fusion (lacking the SET domain) did not result in comparable Hoxa9 promoter modifications despite overexpression of the Hoxa9 transcript (7, 8). Transductions using the MLL–eleven nineteen leukemia and MLL–FK506-binding protein fusions demonstrated binding to HoxA promoters and increases in H3 acetylation but no corresponding increases in H3 Lys4 methylation (36). These experiments suggest distinct mechanisms of action in which Hoxa9 is positively regulated by different MLL gene fusions. In addition, our ChIP experiments showed a correlation between the overexpression of Hoxa7 and Hoxa9 and a decrease in the repression-associated methylation of histone H3 Lys9 at each promoter; however, the domain and/or complex within the Mll PTD that is associated with this epigenetic modification is unknown. Interestingly, we did not find similar histone modifications at the Hoxa10 promoter, suggesting its mechanism of overexpression is distinct from those we observed in Hoxa7 and Hoxa9 in MllPTD/WT mice.
Exactly how the associated aberrant epigenetic modifications come about is unknown but must be directly or indirectly related to the PTD itself. Several possible mechanisms are now conceivable with the recent evidence that WT MLL directly binds to the promoter of these HoxA target genes, together with its supercomplex of repressing and activating proteins (7, 8). The duplication of the DNA-binding AT-hook DNA-binding domain within the Mll PTD may have conferred sustained and therefore excessive binding and increased activation at the relevant Hoxa7 and Hoxa9 promoters or could in some way enhance the recruitment of the Mll PTD and its supercomplex to its target genes (37). Alternatively, the associated supercomplex of activating and repressing components may be altered by the PTD in a way that changes a critical balance between these various complexes, excessively favoring target gene activation. It is conceivable that the binding of the Mll PTD to the promoter of these HoxA genes may interfere with the recruitment of the repression machinery. Careful biochemical characterization of these regulatory regions within the promoters of downstream targets of Mll will provide further insights into the dynamic regulation of these gene products in vivo.
Retroviral overexpression of Hox genes, including HoxA members, has been associated with massive deregulation of hematopoiesis and leukemia (27). Despite the sometimes substantial overexpression of the same HoxA genes observed in our MllPTD/WT mice, they displayed no signs of malignant transformation at 2 years of age. The increase in MllPTD/WT progenitor cell number and proliferation during CFU and liquid culture assays, respectively, suggests that HoxA overexpression exerts its effect on immature cells of the hematopoietic compartment but by itself is insufficient to cause leukemic transformation. Overexpression of Hoxa7 or Hoxa9 rapidly induces AML in mice but requires forced coexpression of the Hox cofactor Meis1 (16). MllPTD/WT cells overexpressing Hoxa7 and Hoxa9 genes in our study did not display any increase in Meis1 transcript. Further, AML patients harboring the MLL PTD also undergo epigenetic silencing of their WT MLL allele in their leukemic blasts that contributes to enhanced cell survival (13). Our Mll PTD mice express the WT Mll allele, presumably because this later event requires cooperation with other alterations. Finally, the MLL PTD has been shown to occur simultaneously with tyrosine kinase mutations in AML, such as the FLT3 ITD (38) and Rhe-PDGFR fusion (39), neither of which has been detected in our MllPTD/WT mice. Thus, the failure of our mice to develop frank leukemia is likely related to the absence of additional molecular aberrations that are critical for malignant transformation to occur in the presence of the Mll PTD.
While it would seem most logical that we are observing the earliest events in a continuum of how the Mll PTD contributes to the leukemic phenotype, it must also be considered that some or all of the abnormalities we have observed may not be related to the leukemogenic process as it occurs in patients with AML and the MLL PTD. For example, while the excessive production of erythroid and megakaryocytic progenitor populations is consistent with the specific role of Hoxa10 in these lineages (28) and with Hoxa10 overexpression found in the MllPTD/WT mice, AML patients with the MLL PTD rarely have blasts of erythroid or megakaryocyte lineage. Clearly, additional work in combination with mice harboring other genetic defects, as described above, will need to be undertaken in order to shed light on the role of the Mll PTD in the genesis of leukemia.
In summary, we have engineered a mouse with the Mll PTD driven off of its endogenous promoter. We provide what we believe to be the first in vivo evidence for a mechanism by which an Mll gene fusion (in this case, a self fusion) strongly associated with AML leads to aberrant target gene activation, i.e., increased histone H3/H4 acetylation and histone H3 Lys4 methylation.
Methods
All primers and primer/probe sets used, along with conditions of amplification can be found in Supplemental Tables 1 and 2.
Development of the MllPTD/WT mouse. A P1 genomic clone containing the murine Mll genomic fragment was obtained from Incyte (formerly Genome Systems). This was subcloned as 2 fragments (F1 and F2) into pBluescript II as BamHI fragments. The BamHI/SmeI fragment from F2 was then subcloned into the BamHI and SmeI sites of ploxPneo1. F1 was then subcloned into the BamHI site of ploxPneo containing F2. This generated a targeting vector with ploxPneo1 as the vector backbone and genomic Mll sequences spanning intron 4 through intron 11 (Figure 1). All animal research was reviewed and approved by the Institutional Laboratory Animal Care and Use Committee at the Ohio State University.
Approximately 60 μg of the targeting vector was linearized within intron 4 of Mll using the unique SfiI restriction site and introduced into 3 x 107 R1 ES cells. A plasmid vector expressing Cre recombinase from a CMV promoter (pCre) that conferred puromycin resistance was transiently transfected into C186 MllPTD/WT ES cells by electroporation. Cells were cultured overnight and then transferred to a selective media containing 1 μg/ml of puromycin, grown further, and subcloned. Following transient transfection, selection, and expansion, DNA-PCR was performed using an upstream primer from intron 11 and a downstream primer from the ploxPneo vector. This amplified a 0.9-kb band, suggesting that the pgk-neo cassette had been removed, and this was confirmed by sequencing. Next, 2 independently targeted ES cell clones were selected for generating chimeric mice. After verifying that these clones were karyotypically normal and free of mycoplasma contamination, 15 ES cells from each clone were injected into 3.5-day-old blastocysts from pregnant C57BL/6 female mice (The Jackson Laboratory). Next, the chimeric embryos were transferred into pseudopregnant recipient Swiss-Webster female mice. Micromanipulations, blastocyst transfers, and generation of chimeric mice were performed at the Transgenic Shared Resource of The Ohio State University Comprehensive Cancer Center. Backcrossing to a pure C57BL/6 background was carried out in 5 generations using marker-assisted breeding (speed congenics) (The Jackson Laboratory).
Southern blot analysis. High molecular weight DNA extracted from cells was digested with SbfI and NdeI (New England Biolabs Inc.), and Southern blotting was performed according to previously described techniques (40). DNA was transferred to Hybond N+ nitrocellulose membrane (Amersham Biosciences) and hybridized to a cDNA fragment spanning the Mll intron 4–exon 5 junction. The predicted fragment size for the Mll PTD allele was 40 kb while the predicted fragment size for the WT Mll allele was 18 kb.
Genotyping. DNA was prepared from tail biopsies of adult and newborn mice using standard phenol/chloroform extraction. DNA PCR was performed using a forward primer located within intron 6 and a reverse primer located within the targeting vector backbone that amplifies a 900-bp product. Total RNA was extracted from tissues using the RNeasy Mini Kit (Qiagen), and RT-PCR was performed using MMLV Reverse Transcriptase (Invitrogen) with a forward primer located within exon 11 and a reverse primer located within exon 6, which resulted in the generation of a 335-bp PCR product.
CFU progenitor assays and liquid stem cell cultures. Splenocytes were isolated from MllPTD/WT, MllWT/WT, and Mllaf9/WT mice (generously provided by T. Rabbitts, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, via J. Kersey, Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA). Single-cell suspensions were prepared in Iscove’s Modified Dulbecco’s Medium (Invitrogen) containing 2% of FBS (HyClone) from n = 6–8 mice per genotype. Cells were then plated at a density of 2 x 104 cells/dish in M3434 methylcellulose (StemCell Technologies) to assess splenic progenitor populations. All colony assays were performed according to the manufacturer’s protocol (StemCell Technologies). Secondary replating assays were enumerated and performed on day 14 of primary cultures. Cells were replated at a density of 104 cells/dish in M3434 methylcellulose (StemCell Technologies). Secondary cultures were also performed in liquid medium RPMI 1640 with GlutaMAX (Invitrogen) plus 100 ng/ml of stem cell factor, 10 ng/ml of mouse IL-3, and 20 ng/ml of human IL-6 (StemCell Technologies) and seeded simultaneously from primary CFU progenitor assays and in parallel with secondary replating assays at a density of 104 cells/ml.
ChIP. ChIP assays were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate USA Inc.). Approximately 3 x 106 cells were used per assay, and approximately 1% of the extracted chromatin solution was saved for use as input DNA. Immunoprecipitation with the following antibodies was performed: anti-acetyl-Histone H4, anti-acetyl-Histone H3, anti-dimethyl-Histone H3 Lys4, and anti-dimethyl-Histone H3 Lys9 (Upstate USA Inc.). The DNA was extracted using phenol-chloroform, precipitated with ethanol, and dissolved in water. Immunoprecipitations were analyzed by nested PCR using GeneAmp PCR System 9700 (Applied Biosystems). The cycle number and the amount of template were optimized to ensure that results were within the linear range of amplification. The PCR products were size fractionated through 1.0% agarose/ethidium bromide gels.
Quantification of histone acetylation and methylation at Hoxa7 and Hoxa9 promoters in MllPTD/WT, MllWT/– (generously provided by the late S. Korsmeyer), and MllWT/WT DNA extracted from spleen and BM cells was undertaken using real-time quantitative PCR with SYBR green incorporation (Applied Biosystems). Primers were designed and optimized to amplify genomic sequences in the Hoxa7 and Hoxa9 promoters (Supplemental Tables 1 and 2). Each reaction used 2 μl of DNA template and 12.5 μl SYBR Green PCR Master Mix (Applied Biosystems) and was normalized to input DNA. The specificity of PCR products was analyzed by addition of a melting curve cycle, which consisted of 1 cycle each of 60°C for 15 seconds, 95°C for 20 seconds, and 60°C for 15 seconds, followed by analysis on Dissociation Curve Analysis software, version 1.0 (Applied Biosystems).
Comparative real-time RT-PCR. Total RNA was extracted from mouse BM, spleen, and peripheral blood samples and cDNA prepared as previously described (41). Primer and probe sets to detect murine Hoxa7, Hoxa9, and Hoxa10 transcripts were designed using Primer Express (Applied Biosystems). Real-time RT-PCR efficiency was optimized for each primer/probe set using established guidelines (User Bulletin, Applied Biosystems; http://www.appliedbiosystems.com/support/apptech/). Comparative real-time RT-PCR was performed following the manufacturer’s suggested procedures and using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). To normalize for RNA content, the r18s primer/probe set was included in multiplex reactions. Data were analyzed using SDS version 1.7a software (Applied Biosystems) and reported as 2–Ct, which represents the fold differences when normalized to an internal control (r18s) and then compared with MllWT/WT sibling control samples analyzed on the same 96-well plate. For cell-sorting real-time RT-PCR experiments, whole BM cells were harvested and stained with antibodies to Ter119, CD71, Gr-1, CD11b, B220, and CD3. A minimum of 1 x 105 cells positive for each lineage marker was isolated using a FACSVantage (BD). Equivalent numbers of cells positive for each marker from MllWT/WT and MllPTD/WT were used to quantify HoxA gene expression. Real-time RT-PCR was then performed as described above. We used our previously published methodology to quantify absolute levels of Mll PTD and Mll WT transcripts present in BM, spleen, and blood of MllPTD/WT mice by real-time RT-PCR (13). Further information on primers and probes is provided in Supplemental Methods.
Flow cytometric analysis for lineage and subset determination, proliferation, and apoptosis. Cell lineages and hematopoietic subsets within the BM, spleen, and peripheral blood were assessed using lineage-specific antibodies as previously described (15, 41). Immunofluorescent analysis of antigen expression was performed using a FACSCalibur (BD) and CellQuest software (version 3.3; BD). For quantitative assessment of cellular proliferation, BrdU incorporation was measured according to the manufacturer’s protocol (BD Biosciences — Pharmingen). To assess differences in apoptosis, annexin V and PI staining were used and performed according to manufacturer’s protocol (BD Biosciences — Pharmingen).
Whole mount in situ. Whole mount in situ was performed on E12.5 embryos as previously described (1), using digoxygenin-labeled riboprobes generated according to manufacturer’s protocols (Roche Diagnostics). Primer pairs used to generate both sense and anti-sense probes first standardized on WT embryos can be obtained in Supplemental Tables 1 and 2.
Skeletal analysis. Intact skeletons were prepared using sex-matched MllPTD/WT and MllWT/WT mice 20 weeks of age using previously described methods (42). The microCT images were performed as follows: the sacral vertebrae specimens were mounted in 50-ml plastic test tubes filled with glycerol and imaged at 32 kV, 1 mA, and 1-second exposure with image intensifier operating in 7-inch mode. The spatial resolution of the images is 60 μm.
Statistics. A Wilcoxon’s signed-rank test was used to compare differences in CFU growth between the MllWT/WT and MllPTD/WT splenocytes. The Hoxa7, Hoxa9, and Hoxa10 expression levels for each tissue were compared between MllWT/WT and MllPTD/WT mice using a 2-sample 2-tailed Student’s t test.
Acknowledgments
This work was funded in part by the Lady Tata Memorial Trust Award (to A.M. Dorrance) and National Cancer Institute grant funding (R01 CA89341 to M.A. Caligiuri, K01 CA96887 to S.P. Whitman, and K08 CA089317 to L.J. Rush). The microCT images were provided by Kim Powell at the Cleveland Clinic Musculoskeletal Imaging Core, funded in part by a National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Core Center grant (P30 AR050953-01). We thank Jay Hess for kindly providing the Meis1 primer/probe set for quantification of Meis1 transcript, Terry Rabbitts and John Kersey for providing the Mllaf9/WT mice, the late Stanley Korsmeyer for providing the MllWT/– mice, and the staff of the Transgenic Animal, DNA, and Mouse Phenotyping Shared Resources at The Ohio State University Comprehensive Cancer Center for their assistance.
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