Regulation Of Hox Gene Expression Research Articles

The regulation of Hox gene expression during animal development

Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.

The structure of Polycomb-repressed chromatin in the bithorax complex

The 300 kb of the Drosophila bithorax complex (BX-C) is the original model for studying Hox gene repression by Polycomb group proteins. Decades of genetic experiments have led to the hypothesis that Polycomb organizes BX-C chromatin differently in each of the abdominal parasegments, and the structure of the chromatin is critical for maintaining cell identity. Studying the molecular organization of chromatin in individual parasegments has been technically difficult. We solved this problem by developing a sorted nuclei ChIP-seq pipeline. In this system, transgenic embryos produce tagged nuclei in single parasegments. Using FACS, we sorted these tagged nuclei and performed small-scale ChIP-seq. Initial results show that a histone mark catalyzed by Polycomb group proteins, trimethylated histone H3 at lysine 27 (H3K27me3), covers less of the BX-C as we move from PS5 to PS7. The boundaries of the H3K27 methylation correlate precisely with previously identified CTCF binding sites, a protein known to act as a chromatin barrier. Correspondingly, a mark that correlates with gene activation, H3K4me3, appears over the transcription start sites of BX-C genes that are not associated with Polycomb histone methyltransferase activity. Going forward, we will assay the localization of Polycomb group proteins and many other chromatin regulatory proteins to gain a complete picture of the chromatin environment that regulates Hox gene expression in the developing embryo. Still, the current results offer a compelling first look at BX-C chromatin in vivo, and provide molecular support to a long-standing genetic hypothesis.

Spatio-temporal expression ofHOXgenes in human hindgut development

Hox genes belong to a highly conserved subgroup of the homeobox gene superfamily. Studies of animal models have emphasized their role in defining the body plan by their coordinated expression along the body axis during ontogeny. Although an important role of HOX genes in human development is assumed, little is known about their expression during human ontogenesis. Therefore, we investigated the expression of the nine most posterior members of the HOXA, HOXB, HOXC, and HOXD clusters in embryonic hindgut between weeks 6 to 12 and in adult rectal tissue. Applying in situ hybridization and immunohistochemistry, we observed expression of HOXA11, HOXA13, HOXD12, and HOXD13 in developmental week 6. However, expression of HOXD12 faded during weeks 7 and 8, and then became increasingly re-expressed during week 9 in humans. With the exception of HOXD13, all expressed HOX genes dropped below detection limits in week 11. Adult rectal tissue displayed distinct HOXA11, HOXA13, HOXD12, and HOXD13 expression patterns within the rectal layers. Our data suggest a strict spatio-temporal regulation of HOX gene expression during human development, supporting the idea of their role as key regulators. Nonetheless, the expression pattern of distinct HOX genes differs markedly from animal models.

Transcription Regulation of HOX Genes in Normal Hematopoiesis and Leukemogenesis in Children

AbstractAbstract 4614 Introduction:The homeodomain genes (HOX genes) encode a family of highly conserved transcription factors that play fundamental roles during embryogenesis. HOX genes are also important regulators in hematopoiesis. In leukemogenesis, dysregulated expression of HOX genes has been found. Despite many correlative studies, the mechanism of establishment of leukemia specific HOX gene expression patterns in hematopoietic cells remains to be elucidated.Histone methylases and demethylases (Trithorax (TrxG), JMJD3 and Polycomb-group (PcG) genes) are chromatin modifiers regulating global gene expression through chromatin remodeling in many biological processes. PcG genes can also interact with DNA methyltransferases and alter their activity. Our previously published data showed that HOX gene expression correlated with the level of DNA methylation. These data together with the stabilizing function of PcG genes on HOX expression in embryogenesis suggest the involvement of histone modifiers in the regulation of hematopoietic HOX gene expression. Methods:To investigate the regulation of HOX expression in leukemogenesis, we determined mRNA levels of the representative groups of HOX genes (HOXA, HOXB, CDX1/2), PcG genes (EZH2, BMI1), MLL and demethylases (JMJD3, UTX) in samples of childhood AML (N=41) and healthy controls (N=5). We also studied the dynamics of HOX genes and chromatin modifiers in preleukemic and diagnostic samples of a patient who underwent secondary leukemia.Quantification of gene expression was performed using qPCR assays as previously described. Results:Expression patterns for the majority of HOX genes differed significantly among morphologically defined subgroups of AML with AML M3 having the lowest expression of all HOX genes. Children with AML M5 expressed HOXA cluster at the highest level, while HOXB genes were highly expressed in M5 and M4 subtype. Subgroups defined according to molecular genetics showed similar results. The presence of PML/RARa fusion gene was associated with very low expression of all HOX genes whereas MLL+ and CBFb/MYH11+ patients expressed higher levels of HOXA genes. We also assessed the prognostic significance of particular HOX genes and found that the HOXA cluster was expressed at very low levels in standard risk cases compared to the high risk group (P<0.0001 for most HOXA genes), which is in concordance with previously published results in adult AML (Andreeff et al. 2008).Determination of mRNA levels of histone modifiers showed an overall level of high expression across various AML subgroups. Nevertheless, some were uniformly expressed in AML patients (EZH2, MLL), while others were differentially expressed with the lowest level in the M3 subtype (BMI1, JMJD3). Interestingly, we found a correlation between HOX gene expression and levels of JMJD3, which was mainly evident in CBFb-MYH11+, PML-RARa+ and AML1-ETO+ patients. JMJD3 levels were also correlated with another demethylase, UTX. A positive trend between HOX gene expression and JMJD3 was identified in healthy controls as well.Analysis of the sample from preleukemic period of the patient with secondary leukemia (secALL with MLL translocation) allowed us to study the dynamics of HOX gene expression during leukemogenesis. The diagnostic secALL sample showed an expression pattern of HOX genes typical for MLL+ leukemia. However, the profile of HOX genes in preleukemic sample (16 months before secALL) resembled the pattern found in healthy controls. Nonetheless, 90% of these seemingly normal hematopoietic cells were confirmed by FISH analysis to carry MLL/FOXO3A. Thus, even though MLL is a well known regulator of HOX genes, there must be an additional mechanism, that establishes the expression pattern of HOX genes typical in MLL+ patients. Conclusion:In summary, we identified different expression patterns of HOX genes in particular subtypes of childhood AML that significantly correlated with prognosis. Our results indicate that histone modifiers JMJD3 and UTX might be involved in the regulation of HOX gene expression. Moreover, these data also suggest that histone demethylases could cooperate with specific genetic aberrations implicated in chromatin remodeling on regulation of HOX genes.The analysis of secondary leukemia suggests that additional alterations are required to deregulate HOX expression in at least some MLL+ patients. Disclosures:No relevant conflicts of interest to declare.

Cdx1 Interacts Physically with a Subset of Hox Proteins

Cdx and Hox gene families encode homeodomain-containing transcription factors involved in anterior-posterior vertebral patterning. Although Cdx proteins are direct transcriptional regulators of Hox gene expression, both Hox and Cdx proteins are known to interact with other homeodomain transcription factors, leading us to speculate that Cdx and Hox proteins may also interact physically. In testing this, we found that that Cdx1 is indeed capable of associating with a subset of Hox proteins. This interaction is localized to the homeodomain region of both classes of proteins, is reliant on specific arginine residues in helix I of the Hox homeodomain, and is further modulated by N-terminal Hox sequences. More promiscuous interactions were seen with Hox proteins expressed in vivo, suggestive of bridging factors or post-translational modifications. Finally, we demonstrate that this interaction modulates Cdx-Hox transcriptional activity on a Hox-responsive element. This study is the first example of Cdx-Hox protein interactions and suggests that such complexes may modulate Hox and/or Cdx function.

Nonstructural protein-1 of respiratory syncytial virus regulates HOX gene expression through interacting with histone

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. In searching for its mechanism, the present study was designed to screen the interacting proteins of two important nonstructural (NS) proteins in human BECs. The subcellular localization and the effects of NS on HOX gene expression were also examined. The results showed that NS1 was distributed throughout the nucleus and cytoplasm, while NS2 was mainly distributed in cytoplasm of BECs. NS1 interacted specifically with host histone H2BD, inducing histone ubiquitination and subsequent HOX gene expression. In conclusion, the results of the present study indicated that RSV NS-1 induces HOX gene expression, through histone ubiquitination in a BEC cell line, which may provide a novel conception for understanding the relationship between severe RSV bronchiolitis during early life and the development of subsequent asthma.

Reduction of Hox Gene Expression by Histone H1 Depletion

The evolutionarily conserved homeotic (Hox) genes are organized in clusters and expressed collinearly to specify body patterning during embryonic development. Chromatin reorganization and decompaction are intimately connected with Hox gene activation. Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. To investigate the potential role of H1 and higher order chromatin folding in the regulation of Hox gene expression, we systematically analyzed the expression of all 39 Hox genes in triple H1 null mouse embryos and ESCs by quantitative RT-PCR. Surprisingly, we find that H1 depletion causes significant reduction in the expression of a broad range of Hox genes in embryos and ESCs. To examine if any of the three H1 subtypes (H1c, H1d and H1e) is responsible for decreased expression of Hox gene in triple-H1 null ESCs, we derived and characterized H1c−/−, H1d−/−, and H1e−/− single-H1 null ESCs. We show that deletion of individual H1 subtypes results in down-regulation of specific Hox genes in ESCs. Finally we demonstrate that, in triple-H1- and single-H1- null ESCs, the levels of H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) were affected at specific Hox genes with decreased expression. Our data demonstrate that marked reduction in total H1 levels causes significant reduction in both expression and the level of active histone mark H3K4me3 at many Hox genes and that individual H1 subtypes may also contribute to the regulation of specific Hox gene expression. We suggest possible mechanisms for such an unexpected role of histone H1 in Hox gene regulation.

Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Dual Function of Histone H3 Lysine 36 Methyltransferase ASH1 in Regulation of Hox Gene Expression

Hox genes play important roles in haematopoietic development in mammals. ASH1 is a member of the trithorax group (trxG) that is required for proper expression of Hox genes and is preferentially expressed in haematopoietic stem cells. We have recently reported that ASH1 methylates histone H3 at lysine 36 (K36) but its biological function has remained elusive. Here we show that ASH1 regulates Hox gene expression positively and negatively in a leukemic cell line K562 and is required for myelomonocytic differentiation of murine haematopoietic stem cells. ASH1 binds to endogenous Hox loci in K562 cells and its knockdown causes reduced expression of Hox genes. In addition, ASH1 and MLL1 induce more than 100-fold activation of Hox promoters in HeLa cells if expressed simultaneously but not individually. Notably, ASH1 harbouring a point mutation that kills methyltransferase activity is more efficient than wild type ASH1 in Hox gene activation, indicating that K36 methylation is not a prerequisite for Hox gene expression. Moreover, tethering wild type or catalytically inactive methyltransferase domain of ASH1 to a heterologous promoter causes downregulation or upregulation, respectively, of transcription, supporting a hypothesis that K36 methylation imparts repression. Knockdown of ASH1 in K562 cells in vitro causes increased expression of ε-globin gene and reduced expression of myelomonocytic markers GPIIb and GPIIIa, whereas knockdown of ASH1 in murine haematopoietic stem cells in vivo results in decreased number of macrophages and granulocytes, a phenotype similar to that induced by loss of mll1 function. Taken together, our data suggest that ASH1 and MLL1 synergize in activation of Hox genes and thereby regulate development of myelomonocytic lineages from haematopoietic stem cells.

Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

Crosstalk between leukemia-associated proteins MOZ and MLL regulates HOX gene expression in human cord blood CD34+ cells

MOZ and MLL, encoding a histone acetyltransferase (HAT) and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. In MOZ (MOnocytic leukemia Zinc-finger protein)/CBP- or mixed lineage leukemia (MLL)-rearranged leukemias, abnormal levels of HOX transcription factors have been found to be critical for leukemogenesis. We show that MOZ and MLL cooperate to regulate these key genes in human cord blood CD34+ cells. These chromatin-modifying enzymes interact, colocalize and functionally cooperate, and both are recruited to multiple HOX promoters. We also found that WDR5, an adaptor protein essential for lysine 4 trimethylation of histone H3 (H3K4me3) by MLL, colocalizes and interacts with MOZ. We detected the binding of the HAT MOZ to H3K4me3, thus linking histone methylation to acetylation. In CD34+ cells, depletion of MLL causes release of MOZ from HOX promoters, which is correlated to defective histone activation marks, leading to repression of HOX gene expression and alteration of commitment of CD34+ cells into myeloid progenitors. Thus, our results unveil the role of the interaction between MOZ and MLL in CD34+ cells in which both proteins have a critical role in hematopoietic cell-fate decision, suggesting a new molecular mechanism by which MOZ or MLL deregulation leads to leukemogenesis.

Huntingtin facilitates polycomb repressive complex 2

Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat α-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes. Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large α-helical domains. Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9. Supporting a direct stimulatory role, full-length recombinant huntingtin significantly increased the histone H3K27 tri-methylase activity of reconstituted PRC2 in vitro, and structure–function analysis demonstrated that the polyglutamine region augmented full-length huntingtin PRC2 stimulation, both in HdhQ111 EBs and in vitro, with reconstituted PRC2. Knowledge of full-length huntingtin's α-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

Cdx2 regulation of posterior development through non-Hox targets

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively.

The Polycomb Complex Protein mes-2/E(z) Promotes the Transition from Developmental Plasticity to Differentiation in C. elegans Embryos

We have used expression profiling and in vivo imaging to characterize Caenorhabditis elegans embryos as they transit from a developmentally plastic state to the onset of differentiation. Normally, this transition is accompanied by activation of developmental regulators and differentiation genes, downregulation of early-expressed genes, and large-scale reorganization of chromatin. We find that loss of plasticity and differentiation onset depends on the Polycomb complex protein mes-2/E(Z). mes-2 mutants display prolonged developmental plasticity in response to heterologous developmental regulators. Early-expressed genes remain active, differentiation genes fail to reach wild-type levels, and chromatin retains a decompacted morphology in mes-2 mutants. By contrast, loss of the developmental regulators pha-4/FoxA or end-1/GATA does not prolong plasticity. This study establishes a model by which to analyze developmental plasticity within an intact embryo. mes-2 orchestrates large-scale changes in chromatin organization and gene expression to promote the timely loss of developmental plasticity. Our findings indicate that loss of plasticity can be uncoupled from cell fate specification.

Cdx1 and Cdx2 are functionally equivalent in vertebral patterning

The Cdx transcription factors regulate anterior–posterior (AP) vertebral patterning, at least in part, through direct regulation of Hox gene expression. Analysis of allelic series of Cdx mutant mice suggests functional overlap between these family members. However, the lack of a Cdx2 null mutant makes these analyses incomplete. Moreover, Hox proteins are sometimes redundant, making it difficult to discern whether Cdx members regulate identical Hox target genes in a redundant manner, or whether they regulate separate Hox genes which then converge on events related to vertebral patterning. To more directly assess this question, we developed a “knock in” model whereby Cdx2 was substituted for Cdx1. Consistent with functional redundancy Cdx2 “knock-in” mice exhibited perfect complementation of the Cdx1-null phenotype, as evidenced by the lack of skeletal defects or altered expression of Hox genes typically impacted by Cdx1 loss-of-function.It has been proposed that vertebral AP patterning is reliant on a gradient of the sum total of Cdx proteins, a posit that is consistent with functional redundancy between Cdx family members. To further assess this, we generated a gain-of-function model using BAC trangenesis to alter Cdx1 dosage. Cdx1 BAC transgenic mice overexpressed Cdx1 mRNA and protein, and fully complemented the Cdx1 null allele. However, gain of Cdx1 dosage via this BAC transgene in an otherwise wild type background had no discernible effects on vertebral patterning or Hox gene expression, suggesting that a moderate alteration in the Cdx protein gradient is of no consequence.

Regulatory noncoding RNAs atHoxlociThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB’s 51st Annual Meeting – Epigenetics and Chromatin Dynamics, and has undergone the Journal’s usual peer review process.

There is growing awareness of the importance of noncoding (nc)RNAs in the regulation of gene expression during pattern formation in development. Spatial regulation of Hox gene expression in development controls positional identity along the antero-posterior axis. In this review, we will focus on the role of short ncRNAs that repress Hox genes in Drosophila and mammals by RNA interference (RNAi), on long ncRNAs that may repress a Hox in cis in Drosophila by transcriptional interference, and on a novel long ncRNA that functions in trans to regulate Hox genes mammals.

Cdx4 Differs from Cdx2 in Its Oncogenic Potential and Its Regulation of Hox Gene Expression in the Murine Bone Marrow Transplantation Model.

Cdx4 is known to be of importance for specification of cell fate in embryonic hematopoiesis with defects leading to severe perturbation of blood formation. When overexpressed in a murine hematopoietic stem cell line, Cdx4 is capable to enhance progenitor formation in vitro and promote lymphoid reconstitution of lethally irradiated, transplanted mice in vivo.In line with this important function of Cdx4 in early hematopoiesis, we analyzed expression of Cdx4 in highly purified subpopulations isolated from murine bone marrow (BM) cells by TaqMan qPCR. Cdx4 showed an expression profile known from other stem cell regulatory genes with high expression in early hematopoietic progenitors followed by decreasing expression towards the more differentiated stages of hematopoiesis, with a more than 1200-fold lower expression in total BM cells compared to progenitor enriched 5-FU BM cells (n=3).To test the impact of Cdx4 on murine progenitors, we retrovirally transduced 5-FU BM cells with Cdx4. Overexpression of Cdx4 induced growth of BM cells in liquid expansion assay (Cdx4 5.7×108±2.2×108 SEM, EGFP 2.6×106±9×105 SEM, p=0.020; cell numbers after 14 days in cytokine supplemented medium, n=5). In addition, expression of Cdx4 conferred serial replating capacity to murine BM progenitors compared to empty vector control (CFU total after 3rd replating: 4.5×109±1.3×109 SEM/500 input cells in 1st CFC, n=5). This effect was significantly stronger compared to hematopoietic progenitors overexpressing the leukemogenic Cdx2 (p=0.008). Immunophenotyping of cells after 3rd replating showed expression of mainly myeloid antigens and cytospin preparation revealed a mature myeloid morphology. Interestingly, these colonies were able to engraft lethally irradiated mice and showed multilineage engraftment (lymphoid:myloid ratio week 16 after transplantation: 0.5:1, n=2), indicating the ability of Cdx4 expressing colonies to maintain stem cell properties in vitro.In contrast to Cdx2-transplanted mice which showed a severe myeloid bias, regular peripheral blood analysis of mice transplanted with Cdx4 overexpressing BM cells showed multilineage engraftment confirmed by immunophenotyping and normal hematological parameters (RBC 6.7×109±4.2×108, WBC 5.8×106±5.19×105; lymphoid:myeloid ratio 1.4:1; week 8–28). Of note, with a median latency of 309 days after transplantation, nine out of ten mice transplanted with Cdx4-transduced BM cells died of transplantable leukemia. In six out of seven cases we found single retroviral integration sites, indicating a monoclonal origin of the disease. We could determine three different integration sites located between 200 and 700 bp upstream of coding sequences (n=4; Opa3, Akap1, Sema4d). The integration sites of two other mice were located intragenic (Zfyve2, Zfp407), indicating that insertional mutagenesis might be a necessary factor for Cdx4 induced leukemogenesis. Moreover, qRT-PCR revealed that Cdx4 in contrast to Cdx2 did not induce ectopic expression of the leukemogenic Hoxb8 and was associated with a significant lower (7.8-fold) expression of the leukemogenic Hoxb6 in transduced murine BM cells.Taken together, these data indicate that Cdx4 plays a major role in the regulation of early hematopoiesis. Its expression profile and its hematopoietic activity in different hematopoietic assays clearly differs from Cdx2, which was shown to be highly leukemogenic in mice and to be ectopically expressed in human AML. Murine models analyzing the impact of Cdx4 and Cdx2 expression on hematopoietic development will help to delineate critical differences between the two related genes.

Deregulated Expression of Ubiquitin-Specific Peptidase Genes in Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by mutations in genes regulating transcription and in genes involved in signaling pathways, leading to aberrant cellular differentiation and proliferation. Transcription factors such as RUNX1, the DNA-binding subunit of the core-binding factor, are frequently rearranged in AML by chromosomal translocations. We have characterized a second case of t(7;21) (p22;q22) translocation in AML cells and confirmed that USP42 is a recurrent fusion partner of RUNX1. A 10-fold upregulation of USP42 mRNA was detected in the t(7;21) leukemic cells. The USP42 gene is a member of the ubiquitin-specific peptidase (USP) family which catalyzes the removal of an ubiquitin moiety on target proteins. Targets of USP42 are currently unknown. USPs are deubiquitinating enzymes involved in a growing number of cancers but the role of this family of proteins in leukemogenesis remains to be studied.We have analyzed the expression profile of selected USP genes including USP7, USP14, USP16, USP28 and USP42 in myeloid leukemic samples with different morphologic and karyotypic features by real-time quantitative PCR analysis. USP7, an essential component of the p53-Mdm2 pathway, is a deubiquitinating enzyme for both Mdm2 and p53, involved in cellular proliferation and apoptosis. USP7 regulates p53 stability in a highly complex manner. Different expression profiles of USP7 have been described in cancers. A 2 to 10-fold dowregulation of USP7 was observed in 18 out of 115 (~15%) specimens of myeloid leukemia. Interestingly, we also found a 6 to 38-fold upregulation of USP7 in 9 out of 115 (~8%) leukemic specimens. Four out of five samples of chronic myeloid leukemia in blastic phase expressed high USP7 mRNA level. Involved in DNA repair and in DNA damage-induced apoptosis, USP28 was recently described as a deubiquitinating enzyme of MYC, a highly relevant gene in hematologic malignancies. In response to DNA damage, MYC levels decline following USP28 dissociation from the ubiquitin ligase FBW7. Downregulation of USP28 mRNA expression (3 to 20-fold) was detected in 6 AML specimens with different karyotypes including trisomy 8, complex and normal karyotypes. Two other USPs were also studied because of their potential relevance to hematologic cancers. USP16 regulates HOX gene expression through histone H2A deubiquitination (Joo HY, Nature 2007) and USP14 is highly expressed in myeloid and lymphoid leukemic cell lines (Ishiwata S, J.Biochem 2001). The expression level of these two USPs was not abnormal in our study, but we are currently investigating more leukemic samples to confirm these results.The deregulation of USP7 and USP28 expression and the presence of a recurrent translocation involving USP42 in our study, suggest an important role for this family of enzymes in the pathogenesis of myeloid leukemia. Deregulated expression of genes that encode enzymes involved in the ubiquitin-proteasome pathway opens new fields of investigation, since these enzymes could potentially be targets for novel therapy in AML.

New molecular concepts and targets in acute myeloid leukemia

Most patients with acute myeloid leukemia treated with chemotherapy relapse. It is increasingly recognized that the cause of chemoresistance and relapse resides within the leukemia stem cell population. Successful eradication of leukemia stem cells would require a comprehensive profile of both the acquired molecular lesions and intrinsic features of leukemia stem cells. This review describes recent work identifying molecular markers that may lead to development of novel therapeutics, ultimately aiming to eradicate leukemia stem cells in acute myeloid leukemia. In recent years, novel specific cell surface antigens have allowed identification of leukemia stem cells and permitted their distinction from normal hematopoietic stem cells. Novel concepts of leukemia stem cells and niche interaction have elucidated the mechanisms that control leukemia stem cell survival and chemoresistance. Recent detection of genetic aberrations affecting regulators of HOX gene expression and chromatin modifying enzymes, such as CDX2 and hDOT1L, respectively, elucidates new key players in stem cell self-renewal and leukemic transformation. The discovery of novel markers and survival pathways for leukemia stem cells has increased our potential to specifically target and eliminate the leukemic stem cell compartment, which is likely to improve clinical outcomes in acute myeloid leukemia.

On the nature of thumbs

Differential Hox gene expression make the thumb special