Regulation Of Hox Gene Expression Research Articles

922. Deriving Tissues from Embryonic Stem Cells

Top of pageAbstract Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult, begging the question of whether ES cells give rise to transplantable hematopoietic stem cells (HSCs) in vitro, and limiting the application of ES cells in experimental and therapeutic models. In an effort to characterize factors that differentiate primitive progenitors derived from yolk sac or ES cells into transplantable HSCs, we have examined the effect of conditional expression of several transgenes linked to leukemogenesis, cytokine signaling, or blood formation in model organisms. These genes include the oncoprotein BCR/ABL, the signal transducer STAT5, the bHLH transcription factor SCL, the homeotic selector gene HoxB4 implicated in self-renewal of definitive HSCs, and Cdx4, an upstream regulator of Hox gene expression. BCR/ABL enables engraftment of animals with hematopoietic cells that exhibit multi-lineage differentiation potential, but leukemia invariably develops. STAT5 activation enhances hematopoietic proliferation in vitro but enables only limited, transient multi-lineage engraftment after transplantation of irradiated mice. Induction of SCL appears to enhance hematopoietic commitment during ES differentiation but does not promote hematopoietic engraftment. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma enables generates transplantable cells with a definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multi-lineage hematopoiesis in primary and secondary recipients. Cdx4 further enhances the capacity for HSC generation from ES cells. We have used HoxB4 modification of ES-derived HSCs to enable therapeutic transplantation with products of ES cells, including a proof-of-principle application of therapeutic cloning to treat an immunodeficient Rag2−/− mouse, and efforts are underway in murine models of thalassemia. Our studies in the blood forming system are laying a foundation for similar studies in germ cell development, and to applications with human ES cells.

Drosophila FACT is Involved in the Regulation of Hox Gene Expression through Physical and Functional Interaction with GAGA Factor

Drosophila FACT is Involved in the Regulation of Hox Gene Expression through Physical and Functional Interaction with GAGA Factor

Mechanisms of Transformation by MLL

Rearrangements of the mixed-lineage leukemia gene MLL1 (MLL, HRX, ALL1), the human homologue of the Drosophila gene trithorax, are associated with aggressive acute leukemias in both children and adults. Transformation by rearranged forms of MLL1, including in-frame fusion proteins, partial tandem duplications, and amplification of MLL1 through upregulation of Hox gene and cofactor expression apparently results in a block in hematopoietic differentiation. MLL1 regulates Hox gene expression via direct promoter binding and histone H3 Lys 4 methylation mediated by the intrinsic methyltransferase activity of the SET domain. Mll1 knockout leads to loss of Hox gene expression, defects in hematopoiesis, and embryonic lethality. A close homologue, MLL2 is amplified in some solid tumors. MLL2 also has histone H3 Lys 4 methyltransferase activity that is dependent on menin, a protein mutated in multiple neoplasia type I (MEN1) and which is required for normal Hox expression. These findings underscore the importance of the MLL histone methyltransferases in development and disease.

Polycomb Group Regulation of Hox Gene Expression in C. elegans

Polycomb group (PcG) chromatin proteins regulate homeotic genes in both animals and plants. In Drosophila and vertebrates, PcG proteins form complexes and maintain early patterns of Hox gene repression, ensuring fidelity of developmental patterning. PcG proteins in C. elegans form a complex and mediate transcriptional silencing in the germline, but no role for the C. elegans PcG homologs in somatic Hox gene regulation has been demonstrated. Surprisingly, we find that the PcG homologs MES-2 [E(Z)] and MES-6 (ESC), along with MES-3, a protein without known homologs, do repress Hox expression in C. elegans. mes mutations cause anteroposterior transformations and disrupt Hox-dependent neuroblast migration. Thus, as in Drosophila, vertebrates, and plants, C. elegans PcG proteins regulate key developmental patterning genes to establish positional identity.

Global Regulation of Hox Gene Expression in C. elegans by a SAM Domain Protein

Polycomb group (PcG)-mediated repression of C. elegans Hox genes has not been demonstrated, and genes homologous to components of one of the PcG complexes (PRC1) have not been identified in the C. elegans genome. We find that a mechanism of general Hox gene repression exists in C. elegans, carried out in part by SOP-2, a protein related to, but not orthologous with, any PcG protein. sop-2 mutations lead to widespread ectopic expression of Hox genes and homeotic transformations. SOP-2 contains a SAM domain, a self-associating protein domain found in other repressors, including a core component of PRC1 and ETS transcription factors. Phylogenetic analysis indicates that this domain is more closely related to those of the ETS family than to those of PcG proteins. The results suggest that global repression of Hox genes has been taken over by a different branch of the SAM domain family during the evolution of nematodes.

Global down-regulation of HOX gene expression in PML-RARα+ acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene.

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.

Coordinated regulation of HOX gene expression in myometrium and uterine leiomyoma

Objective: To measure HOXA10 gene expression in uterine myometrium and leiomyoma throughout the menstrual cycle. Design: HOXA10 gene expression was measured in paired myometrium and leiomyoma from the same uterus. Setting: University medical center. Patient(s): Thirty-one patients with leiomyoma. Intervention(s): Collection of leiomyoma and myometrial tissue during hysterectomy. Main Outcome Measure(s): HOXA10 gene expression was analyzed by Northern analysis and quantified by laser densitometry. Result(s): Both myometrium and leiomyoma continued to express HOXA10 in the adult and menstrual cycle stage-specific regulation was evident in both tissues. During the mid-secretory phase of the menstrual cycle, HOXA10 expression decreased in both leiomyoma and myometrium. At all points in the menstrual cycle HOXA10 expression in leiomyoma was similar to expression in paired myometrium. Conclusion(s): HOXA10 is expressed in both myometrium and leiomyoma. Coordinated regulation of this transcript factor suggests similar gene activation in both leiomyoma and myometrium. Diminished expression of HOXA10, a gene that normally causes differentiation, may allow increased growth resulting in the increased mitosis seen in the secretory phase. The identification of this developmental control gene in myometrium and leiomyoma may lead to a better understanding of the molecular mechanisms responsible for growth and differentiation of these tissues.

Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice.

The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have identified Ring1B, the product of the Ring1B gene (Rnf2 - Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.

Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos.

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

HOXA10 Expression Is Repressed by Progesterone in the Myometrium: Differential Tissue-Specific Regulation of HOX Gene Expression in the Reproductive Tract

HOXA10 Expression Is Repressed by Progesterone in the Myometrium: Differential Tissue-Specific Regulation of HOX Gene Expression in the Reproductive Tract

HOXA10 Expression Is Repressed by Progesterone in the Myometrium: Differential Tissue-Specific Regulation of HOX Gene Expression in the Reproductive Tract1

HOX genes are essential regulators of development in all multicellular organisms, including humans. We have previously shown that HOXA10 is expressed in the developing uterus and later in the adult human endometrium. HOX genes regulate endometrial development in response to sex steroids. Here, we demonstrate that HOXA10 is expressed in the myometrium as well. In situ hybridization reveals abundant HOXA10 expression, and Northern analysis demonstrates differential HOX gene expression in the myometrium throughout the menstrual cycle. HOXA10 expression decreases in the midsecretory phase, coinciding with high serum progesterone levels. Treatment of primary myometrial cell cultures with progesterone decreases HOXA10 expression in vitro-paralleling the expression seen in vivo. Despite the presence of progesterone receptors in the endometrium and myometrium, HOXA10 is differentially regulated in each tissue by progesterone. HOXA10 expression is induced in the stroma and decreased in the myometrium by progesterone. The differential tissue-specific response of this gene in response to progesterone is likely mediated by sex steroid receptor coactivators or corepressors. Decreased myometrial expression of developmental regulatory genes such as HOXA10 in the nonpregnant uterus may dedifferentiate the myometrium and allow growth in preparation for pregnancy.

The novel C. elegans gene sop-3 modulates Wnt signaling to regulate Hox gene expression.

We describe the properties of a new gene, sop-3, that is required for the regulated expression of a C. elegans Hox gene, egl-5, in a postembryonic neuroectodermal cell lineage. Regulated expression of egl-5 in this cell lineage is necessary for development of the sensory rays of the male tail. sop-3 encodes a predicted novel protein of 1475 amino acids without clear homologs in other organisms. However, the sequence contains motifs consisting of homopolymeric runs of amino acids found in several other transcriptional regulators, some of which also act in Hox gene regulatory pathways. The genetic properties of sop-3 are very similar to those of sop-1, which encodes a component of the transcriptional Mediator complex, and mutations in the two genes are synthetic lethal. This suggests that SOP-3 may act at the level of the Mediator complex in regulating transcription initiation. In a sop-3 loss-of-function background, egl-5 is expressed ectopically in lineage branches that normally do not express this gene. Such expression is dependent on the Hox gene mab-5, as it is in branches where egl-5 is normally expressed. Ectopic egl-5 expression is also dependent on the Wnt pathway. Thus, sop-3 contributes to the combinatorial control of egl-5 by blocking egl-5 activation by MAB-5 and the Wnt pathway in inappropriate lineage branches.

Coordinate Regulation of HOX Gene Expression in Myometrium and Uterine Leiomyomas

Objectives: The molecular factors that regulate the development and growth of uterine leiomyomas are still unknown. HOX genes give developmental identity to individual segments of the paramesonephoric duct. Specifically, HOXA10 leads the development of uterine identity and then in the adult regulates endometrial receptivity. In this study we investigated the expression of HOXA10 in adult uterine myometrium and its regulation during the menstrual cycle. We also examine HOXA10 expression and regulation in uterine leiomyomas.

Plzf regulates limb and axial skeletal patterning.

The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145-/- mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145-/- mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145-/- mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

RARbeta mediates the response of Hoxd4 and Hoxb4 to exogenous retinoic acid.

One action of retinoids in development is the regulation of Hox gene expression. Hoxd4 and Hoxb4 expression in the embryonic hindbrain is anteriorized by retinoic acid (RA) treatment of mid-gestation mouse embryos. Here we demonstrate that retinoic acid receptor beta (Rarb) deficient mice present only a partial anteriorization of either Hoxd4 or Hoxb4 in response to RA treatment. Our results strongly suggest that RARbeta is a mediator of their RA-response, and reveal anteroposterior polarity within a single rhombomere (r). Additionally, we generated mice doubly mutated for Hoxd4 and Rarb in an attempt to identify common morphogenetic pathways between these two genes. We conclude that there are no synergistic interactions between Hoxd4 and Rarb in the specification of the cervical vertebrae.

RARβ mediates the response of Hoxd4 and Hoxb4 to exogenous retinoic acid

One action of retinoids in development is the regulation of Hox gene expression. Hoxd4 and Hoxb4 expression in the embryonic hindbrain is anteriorized by retinoic acid (RA) treatment of mid-gestation mouse embryos. Here we demonstrate that retinoic acid receptor beta (Rarb) deficient mice present only a partial anteriorization of either Hoxd4 or Hoxb4 in response to RA treatment. Our results strongly suggest that RARβ is a mediator of their RA-response, and reveal anteroposterior polarity within a single rhombomere (r). Additionally, we generated mice doubly mutated for Hoxd4 and Rarb in an attempt to identify common morphogenetic pathways between these two genes. We conclude that there are no synergistic interactions between Hoxd4 and Rarb in the specification of the cervical vertebrae. Dev Dyn 1999;215:96–107. © 1999 Wiley-Liss, Inc.

Regulating Hox genes

These two papers address the regulation of Hox gene expression in the mouse embryo. Previous work has shown that Fgfr1 null mutations disrupt gastrulation, probably via an effect on cell movement. Partenen et al.1 Partenen J. et al. Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos. Genes Dev. 1998; 12: 2322-2344 Google Scholar have created a series of Fgfr1 alleles leading to less severe phenotypes, which allow them to dissect a potential role for FGF in patterning the anteroposterior (A–P) axis. Hypomorphic mutations, which reduce the level of Fgfr1 transcription, produce posterior truncation of the embryo and homeotic transformations of the vertebrae similar to those seen in certain Hox gene knockouts. This led them to analyse the effect on Hox expression, and they show that the mesoderm expression of Hoxd4, Hoxb4 and Hoxb9 is altered in a manner consistent with the observed vertebral transformations. Hoxd13 expression is also reduced in the limb buds, consistent with defects in distal limb structures. FGF signalling is believed to be transduced through Ras–MAP kinase and PLCγ pathways. Tyrosine 766 of FGFR1 is an autophosphorylation site that is implicated in transduction via PLCγ. Mutation of Y766 produced vertebral transformations in the opposite direction to those caused by hypomorphic mutations, suggesting that this site is normally involved in repression of ligand-dependent FGFR1 function. It is possible that these FGFR1 mutations affect Hox gene expression by altering cell migration or proliferation. Such alterations are not, however, observed in all mutant alleles, suggesting a more direct involvement of FGF in A–P patterning. One possibility is that FGF alters the expression of Cdx genes, vertebrate homologues of Drosophila caudal. Charité et al.2 Charité J. et al. Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements. DeveIopment. 1998; 125: 4349-4358 PubMed Google Scholar have identified CDX-binding sites within an upstream regulatory element that can drive regionally restricted expression from the Hoxb8 promoter. A subfragment containing four CDX-binding sites can partially reproduce this expression pattern, and mutation of the sites abolishes its effect. They show that factors in E8.5 embryos that bind these sites are present in an A–P gradient, consistent with the expression pattern of Cdx1, -2 and -4. Supershift data also suggest that these factors are CDX proteins. Ectopic expression of Cdx4 alters the expression pattern of reporters driven by the CDX-binding element, and of endogenous Hoxb8. These results suggest that, unlike Drosophila caudal, vertebrate CDX factors are directly involved in establishing the early expression of Hox genes. Although Partenen et al.1 Partenen J. et al. Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos. Genes Dev. 1998; 12: 2322-2344 Google Scholar could not detect significant changes in the expression of Cdx1 and -4 in embryos carrying a hypomorphic FGFR1 mutation, they did not rule out their involvement in FGF-mediated regulation of Hox gene expression, as the dynamic pattern of Cdx expression makes it hard to determine whether they are affected.

The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain.

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins- RING1, BMI1, and hPc2-we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3.

The caudal gene codes for a homeodomain transcription factor that is required for normal posterior development in Drosophila. In this study the biological activities of the Xenopus caudal (Cdx) family member Xcad3 are examined. A series of domain-swapping experiments demonstrate that the N-terminus of Xcad3 is necessary for it to activate Hox gene expression and that this function can be replaced by the activation domain from the viral protein VP16. In addition, experiments using an Xcad3 repressor mutant (XcadEn-R), which potently blocks the activity of wild-type Xcad3, are reported. Overexpression of XcadEn-R in embryos inhibits the activation of the same subset of Hox genes that are activated by wild-type Xcad3 and leads to a dramatic disruption of posterior development. We show that Xcad3 is an immediate early target of the FGF signalling pathway and that Xcad3 posteriorizes anterior neural tissue in a similar way to FGF. Furthermore, Xcad3 is required for the activation of Hox genes by FGFs. These data provide strong evidence that Xcad3 is required for normal posterior development and that it regulates the expression of the Hox genes downstream of FGF signalling.