Hox Gene Activation Research Articles

Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex

WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nzeta of Lys4 (H3K4). The role of WDR5 in the MLL1 complex has recently been described as specific recognition of dimethyl-K4 in the context of a histone H3 amino terminus; WDR5 is essential for vertebrate development, Hox gene activation and global H3K4 trimethylation. We report the high-resolution X-ray structures of WDR5 in the unliganded form and complexed with histone H3 peptides having unmodified and mono-, di- and trimethylated K4, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40-repeat fold. Contrary to predictions, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes.

Hyped‐up for Hox in Hyderabad

The EMBO Workshop on Upstream and Downstream of Hox Genes took place in Hyderabad, India, between 14 and 17 December 2005, and was organized by R. Mishra and F. Karch. ![][1] For many years, EMBO workshops dealing with new insights into the regulation and function of Hox genes have been held in Switzerland. However, last year, the EMBO World Programme, together with the Centre for Cellular and Molecular Biology (CCMB), held a workshop entitled Upstream and Downstream of Hox Genes in the CCMB's hometown of Hyderabad, India. Imagine beautiful gardens full of butterflies and palm trees, a delightful guest house neighbouring a state‐of‐the‐art research centre, all in a peaceful oasis surrounded by a pulsating and crowded Indian city. The question at the beginning of the meeting was whether the talks and discussions would match the stunning environment; it was answered rapidly and affirmatively. Hox genes have had an illustrious and curious history. Perhaps one of the most famous images in biology from the past century is the four‐winged mutant of the fruit fly Drosophila melanogaster caused by mutations in the Hox gene Ultrabithorax ( Ubx ). This fascinating mutant fly, discovered and characterized by Edward B. Lewis, has motivated many investigators to join the field to understand how these genes are responsible for such complex changes in morphology. Another highlight of Hox history was Matthew Scott's, William McGinnis's and Walter Gehring's discovery of the homeodomain—a 60‐amino‐acid DNA‐binding motif now known to be a key domain in hundreds of genes throughout the animal, fungal and plant kingdoms. Although a lot has been learned through experiments stemming from these important discoveries, there are still many open questions. Among the questions being debated in the field and at the meeting are: How is the regulation of the Hox genes initiated and maintained? How do Hox proteins … [1]: /embed/graphic-1.gif

Additional Sex Comb-like 1 (ASXL1), in Cooperation with SRC-1, Acts as a Ligand-dependent Coactivator for Retinoic Acid Receptor

Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with retinoic acid receptor (RAR) in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor box (LVMQLL) of ASXL1 and the activation function-2 activation domain (AF-2 AD) core of the RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro glutathione S-transferase pulldown and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pulldown, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1 and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.

Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb

BackgroundPolycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1.ResultsWe show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp11 sex comb phenotypes and enhanced AntpScx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells.ConclusionOur results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

A clock and wavefront mechanism for somite formation

Somitogenesis, the sequential formation of a periodic pattern along the antero-posterior axis of vertebrate embryos, is one of the most obvious examples of the segmental patterning processes that take place during embryogenesis and also one of the major unresolved events in developmental biology. In this article, we develop a mathematical formulation of a new version of the Clock and Wavefront model proposed by Pourquié and co-workers (Dubrulle, J., McGrew, M.J., Pourquié, O., 2001. FGF signalling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232). Dynamic expression of FGF8 in the presomitic mesoderm constitutes the wavefront of determination which sweeps along the body axis interacting as it moves with the segmentation clock to gate cells into somites. We also show that the model can mimic the anomalies formed when progression of the wavefront is disturbed and make some experimental predictions that can be used to test the hypotheses underlying the model.

WDR5 Associates with Histone H3 Methylated at K4 and Is Essential for H3 K4 Methylation and Vertebrate Development

Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.

HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo

AbstractThe HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors, and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis, we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner, pre–B-cell leukemia transcription factor-1 (PBX1). Additionally, we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo, HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4, a region frequently deleted in HOXA9-induced AMLs. In vitro, HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1.

Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Background:MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis.Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter.Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct.Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector.Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. Here, we show that the Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. Our results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons.

Opposing FGF and retinoid pathways: a signalling switch that controls differentiation and patterning onset in the extending vertebrate body axis.

Construction of the trunk/caudal region of the vertebrate embryo involves a set of distinct molecules and processes whose relationships are just coming into focus. In addition to the subdivision of the embryo into head and trunk domains, this "caudalisation" process requires the establishment and maintenance of a stem zone. This sequentially generates caudal tissues over a long period which then undergo differentiation and patterning in the extending body axis. Here we review recent studies that show that changes in the signalling properties of the paraxial mesoderm act as a switch that controls onset of differentiation and pattern in the spinal cord. These findings identify distinct roles for different caudalising factors; in particular, Fibroblast Growth Factor (FGF) inhibits differentiation in the caudal stem zone, while Retinoic acid (RA) provided rostrally by somitic mesoderm is required for neuronal differentiation and establishment of ventral neural pattern. Furthermore, the mutual opposition of FGF and RA pathways controls not only neural differentiation but also mesoderm segmentation and might also underlie the progressive assignment of rostrocaudal identity by regulating Hox gene availability and activation.

A HOXA10 Estrogen Response Element (ERE) is Differentially Regulated by 17 Beta-estradiol and Diethylstilbestrol (DES)

The molecular mechanisms by which estrogens regulate developmental gene expression are poorly understood. While 17 beta-estradiol is normally present at high concentrations in pregnancy, exposure to the estrogen diethylstilbestrol (DES) in utero induces developmental anomalies of the female reproductive tract. HOX gene expression is altered by DES, leading to abnormal Müllerian duct differentiation. The mechanism of ligand-specific regulation of HOX gene expression by estrogens has not been characterized. To elucidate the molecular mechanism underlying ligand-specific estrogen regulation of HOXA10 expression, we characterized regulatory regions of the human HOXA10 gene. We identified an estrogen response element (ERE) in the human HOXA10 gene that mediated differential ligand-specific estrogen-responsive transcriptional activation. Deletional analysis and reporter expression assays identified two EREs, ERE1 and ERE2, each of which drove estrogen-responsive reporter expression in the Ishikawa human uterine endometrial adenocarcinoma cell line. ERE1 drove reporter expression maximally. This ERE bound ERα and ERβ, and formed a complex that included SRC-1, but not CBP, N-CoR or SMRT. HOXA10 ERE1 drove luciferase reporter activity to eightfold the level driven by the consensus ERE in response to estradiol in Ishikawa cells. While most EREs demonstrate similar transcriptional activity in response to DES or estradiol, here estradiol induced four- to sevenfold greater reporter activity than did DES from HOXA10 ERE1. DES did not alter ER or SRC-1 binding to HOXA10 ERE1. HOXA10 ERE1 therefore demonstrated ligand specificity distinct from the consensus ERE, and unrelated to changes in ER or coactivator/corepressor binding. The ligand specificity of the HOXA10 ERE may explain the molecular mechanism by which DES leads to reproductive anomalies; differential ligand-specific activation of HOX genes may be a molecular mechanism by which DES signaling leads to inappropriate HOX expression and to developmental patterning distinct from that induced by estradiol.

The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins.

Transcriptional on and off states of HOX genes and other developmental control genes are maintained by antagonistic regulators encoded by trithorax group (trxG) and Polycomb group (PcG) genes. The trxG proteins Ash1 and hTRX and the PcG repressor E(z) are histone methyltransferases (HMTases) that methylate distinct lysine residues in the N-terminal tail of histone H3. trxG proteins are generally thought to function as activators of HOX genes, but how histone methylation by Ash1 and Trx promotes HOX gene transcription is not clear. Here, we show that in ash1 and trx mutants expression of HOX genes is lost within their normal expression domains, but we find that, contrary to expectation, this expression is restored in ash1 and trx mutants that also lack PcG gene function. Moreover, such trxG PcG double mutants show severe misexpression of HOX genes and, hence, ectopic activation of HOX genes caused by the removal of PcG gene function also occurs in the absence of ash1 and trx function. Together, these results suggest that the Ash1 and Trx HMTases are not "coactivators" required for transcriptional activation of HOX genes, but function specifically as anti-repressors. We propose that histone methylation by Ash1 and Trx is required continuously throughout development to prevent inappropriate PcG silencing of HOX genes in cells in which they must stay transcriptionally active.

Do Higher-Order Chromatin Structure and Nuclear Reorganization Play a Role in Regulating Hox Gene Expression during Development?

expression and contributes to epigenetic inheritance of gene expression states. The best understood examples are the correlations of histone acetylation and methylation to either active transcription or gene repression (Fischle et al. 2003). There is a growing realization that variant histones, such as histones H2A.Z and H3.3, are also critical in demarcating active and silent chromatin (Meneghini et al. 2003; McKittrick et al. 2004). But can all epigenetic information about gene expression be accounted for by chromatin structure at the level of the nucleosome and its modifications, or could poorly understood higher-order chromatin structures and nuclear organization also have a role? To answer this question we have focused here on chromatin and nuclear structure at the murine HoxB locus. Despite decades of analysis of cis-regulatory elements that control Hox gene expression (see, e.g., Marshall et al. 1994; Popperl et al. 1995; Maconochie et al. 1997; Oosterveen et al. 2003), very little is known about the chromatin structure of Hox loci. The correct spatial and temporal regulation of Hox expression is important for anteroposterior patterning of the embryo (Krumlauf 1994). As with the homologous Antennapaedia (ANT) and Bithorax (BX) complexes in Drosophila, the time and place of mammalian Hox gene activation is colinear with the gene order along the chromosome (Kmita and Duboule 2003). Genes at the 3 end of the clusters are expressed earlier, and in more anterior parts of the embryo, than more 5 genes (Fig. 1). Manipulations of the mouse genome that reposition Hox genes within a cluster, for example moving a 3 gene to a more 5 position, have suggested that a transition from an inactive to an active chromatin state is propagated through Hox clusters from 3 to 5 (van der Hoeven et al. 1996; Kondo and Duboule 1999; Roelen et al. 2002). Is the spatiotemporal regulation of Hox loci therefore controlled by, for example, a progressive sweep of histone modification through the complex?

Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana.

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body which consist of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. We examine the expression patterns of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologues of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. We conclude that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. We argue that these segments constitute a unique body region that bears no obvious homology to previously characterised domains of Hox gene activity.

Dimerization contributes to oncogenic activation of MLL chimeras in acute leukemias

MLL is a histone methyltransferase that can be converted into an oncoprotein by acquisition of transcriptional effector domains following heterologous protein fusions with a variety of nuclear transcription factors, cofactors, or chromatin remodeling proteins in acute leukemias. Here we demonstrate an alternative mechanism for activation of MLL following fusions with proteins (AF1p/Eps15 and GAS7) that normally reside in the cytoplasm. The coiled-coil oligomerization domains of these proteins are necessary and sufficient for leukemogenic transformation induced by the respective MLL fusion proteins. Furthermore, homodimerization of MLL by synthetic dimerization modules mimics bona fide MLL fusion proteins resulting in Hox gene activation and enhanced self-renewal of hematopoietic progenitors. Our studies support an oligomerization-dependent mechanism for oncogenic conversion of MLL, presumably in part by recruitment of accessory factors through the dimerized MLL moiety of the chimeric protein.

Vertebrate HoxB gene expression requires DNA replication.

To study the relationship between DNA replication and transcription in vivo, we investigated Hox gene activation in two vertebrate systems: the embryogenesis of Xenopus and the retinoic acid-induced differentiation of pluripotent mouse P19 cells. We show that the first cell cycles following the mid- blastula transition in Xenopus are necessary and sufficient for HoxB activation, whereas later cell cycles are necessary for the correct expression pattern. In P19 cells, HoxB expression requires proliferation, and the entire locus is activated within one cell cycle. Using synchronous cultures, we found that activation of HoxB genes is colinear within a single cell cycle, occurs during S phase and requires S phase. The HoxB locus replicates early, whereas replication is still required for maximal expression later in S phase. Thus, induction of HoxB genes occurs in a DNA replication-dependent manner and requires only one cell cycle. We propose that S-phase remodelling licenses the locus for transcriptional regulation.

Hox genes as synchronized temporal regulators: implications for morphological innovation.

In vertebrates, clusters of Hox genes express in a nested and hierarchical fashion to endow the embryo's segments with discrete identities. Later in development, members of the same gene family are employed again to pattern the limb, intestinal, and reproductive systems. A careful analysis of the morphologies of Hox mutant mice suggests that the genes provide qualitatively different cues during the specification of segments than they do during the development of more recently derived structures. In addition to the regulatory differences noted by others, the activity of Hox genes during specification of the vertebrate metameres in some recent deletion experiments is inconsistent with a role for them as strictly spatial determinants. On the contrary, the phenotypes observed are suggestive of a role for them as elements of a generic time-keeping mechanism. By contrast, the specification of more recent evolutionary structures appears to be more spatial and gene-specific. These differences in role and effect may suggest some simple mechanisms by which the Hox clusters operate, and rules by which gene networks can be diverted to create new structures over the course of evolution. Specific predictions and experiments are proposed.

Hox genes and kidney patterning.

Hox gene activity is essential for proper organization or pattern of the vertebrate body plan and is necessary for organogenesis. Sequence conservation within this family of genes is high yet they are involved in very diverse developmental processes. How this family functions in these processes is a challenging question, but is important for the understanding of renal organogenesis. Multiple Hox genes are expressed in the kidney and mutation in at least one group of paralogous genes results in severe renal defects. Recent studies in mice with targeted Hox gene mutations and in kidney cell lines demonstrate that these genes have evolved to control tissue specific functions through their ability to regulate the expression of renal morphogens. The studies also demonstrate that Hox gene activity is not only restricted by the domain of expression but also by the specificity of the DNA binding homeodomain. Interestingly, these conserved homeodomains are not wholly interchangeable for normal renal organogenesis while they do appear to be interchangeable for axial skeleton development. It is clear that Hox genes regulate important interactions between the ureteric bud and metanephric mesenchyme. Nevertheless, much work remains to define the expression patterns of multiple Hox genes during kidney development, to better determine the functional relationships of the encoded proteins, and to identify additional Hox downstream targets.

MLL Targets SET Domain Methyltransferase Activity to Hox Gene Promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.

Colinearity and non-colinearity in the expression of Hox genes in developing chick skin.

Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. We found that these characteristics apply to several Hox genes expressed in developing chick skin (Hoxb-4, Hoxa-7 and Hoxc-8), and we classed this group of genes as regionally restricted. To our surprise, we found that most of the Hox genes we examined are regionally unrestricted in their expression in the embryonic chick skin. This second group includes the Hoxd genes, Hoxd-4 to Hoxd-13, Hoxa-11 and Hoxc-6. Temporally, the expression of the regionally restricted genes can be observed by E5 within the epidermis, whereas the spatially unrestricted genes are not expressed in the epidermis until E6.25. Unexpectedly, we found that all the unrestricted genes are expressed concomitantly and therefore do not conform to temporal colinearity. Moreover, the dermal expression for both groups occurs later, but maintains the same anteroposterior patterning to that seen previously in the epidermis. During embryonic day 7-8, expression for all genes is up-regulated within the dense dermis whilst being reduced within the inter-bud regions. Later expression within the bud mesenchyme is down-regulated whilst high levels of transcriptional activity are detectable within the epidermal sheath of each feather bud. These results indicate that the transcriptional activity of Hox genes in the developing chick skin could be important during embryonic skin patterning both by providing regionally restricted positional cues, and also by imparting generic signals necessary for feather morphology.