1: Arch Dermatol Res. 2005 Nov 15;:1-5 [Epub ahead of print] The HOXC13-controlled expression of early hair keratin genes in the human hair follicle does not involve TALE proteins MEIS and PREP as cofactors. Jave-Suarez LF, Schweizer J. Section of Normal and Neoplastic Epidermal Differentiation, German Cancer Research Center, A145, Im Neuenheimerfeld 280, 69120, Heidelberg, Germany, j.schweizer@dkfz.de. We previously showed that the homeodomain protein HOXC13 is involved in the expression control of the early human hair keratin genes hHa5 and hHa2, which contain specific HOXC13 binding sites in their proximal promoters. Hox specificity is generally thought to be enhanced by the interaction with members of the TALE superclass of homeodomain proteins Pbx, Meis, and Prep. Using reverse transcription PCR with total human hair follicle RNA, we demonstrated transcripts of the major TALE proteins PBX1-4, MEIS1, 2 and PREP1, 2 in the human hair follicle. In view of the presence of MEIS/PREP responsive elements in close vicinity to the HOXC13 binding sites of the hHa5 and hHa2 promoters, we determined the expression sites of these TALE proteins in the human hair follicle. We found that MEIS1, MEIS2, PREP1 and PREP2 were differentially expressed in the three layers of the inner root sheath. In addition, MEIS2 and PREP1 exhibited expression in the mid-to upper hair cortex, with PREP1 being also expressed in the dermal papilla and the connective tissue sheath of the hair follicle. In virtually all cases, the expression of these TALE proteins was exclusively cytoplasmic. Considering that in contrast, HOXC13 is expressed in the nuclei of matrix, precortex and lower cuticle cells of the hair follicle, our data suggest that despite the presence of MEIS/PREP binding sites in the hHa5 and hHa2 promoters, the HOXC13-controlled activation of these genes in the hair follicle does not seem to involve these TALE proteins as cofactors. PMID: 16292560 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: Leuk Res. 2005 Oct 7; [Epub ahead of print] Histologic and molecular characterizations of megakaryocytic leukemia in mice. Hao X, Shin MS, Zhou JX, Lee CH, Qi CF, Naghashfar Z, Hartley JW, Fredrickson TN, Ward JM, Morse HC 3rd. Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institute of Health, Rockville, MD 20852, USA. Six cases of megakaryocytic leukemia (MKL) were identified and analyzed for morphology and molecular features. MKL were composed of megakaryocyte lineage cells ranging from immature to quite mature cells. VWF, GATA1 and RUNX1 were strongly expressed in megakaryocytes in both normal spleen and MKL as analyzed by immunohistochemistry (IHC). Altered expression of Meis1, Pbx1 and Psen2 and Lef1 in MKL detected with oligonucleotide microarrays was confirmed by qPCR and IHC. This is the first report of spontaneous MKL in mice, defining VWF as a biomarker for diagnosis and suggesting possible involvement of a series of genes in disease pathogenesis. PMID: 16219351 [PubMed - as supplied by publisher] --------------------------------------------------------------- 3: Int J Dev Biol. 2005;49(7):851-8. PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract. Dintilhac A, Bihan R, Guerrier D, Deschamps S, Bougerie H, Watrin T, Bonnec G, Pellerin I. UMR 6061, Genetique et Developpement, IFR 140, Universite de Rennes 1, Campus Villejean, Rennes, France. While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated. PMID: 16172981 [PubMed - in process] --------------------------------------------------------------- 4: J Biol Chem. 2005 Apr 22;280(16):16484-98. Epub 2005 Feb 18. Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal. Martinez-Ceballos E, Chambon P, Gudas LJ. Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA. Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of the Hoxa1 gene, the most 3' member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1(-/-) embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1(-/-) mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1(-/-) ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1, Postn/Osf2, and the bone sialoprotein gene or BSP), genes that are expressed in the developing brain (e.g. Nnat, Wnt3a, BDNF, RhoB, and Gbx2), and genes involved in various cellular processes (e.g. M-RAS, Sox17, Cdkn2b, LamA1, Col4a1, Foxa2, Foxq1, Klf5, and Igf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1, Oct3/4, Fgf4, and Bmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1(-/-) ES cells express high levels of various endodermal markers, including Gata4 and Dab2, and express much less Fgf5 after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics. PMID: 15722554 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Biol Chem. 2005 Apr 1;280(13):12359-70. Epub 2005 Jan 28. HOXA9 activates transcription of the gene encoding gp91Phox during myeloid differentiation. Bei L, Lu Y, Eklund EA. Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis. PMID: 15681849 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 2005 Mar 18;280(11):10119-27. Epub 2005 Jan 15. MEIS C termini harbor transcriptional activation domains that respond to cell signaling. Huang H, Rastegar M, Bodner C, Goh SL, Rambaldi I, Featherstone M. McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. MEIS proteins form heteromeric DNA-binding complexes with PBX monomers and PBX.HOX heterodimers. We have shown previously that transcriptional activation by PBX.HOX is augmented by either protein kinase A (PKA) or the histone deacetylase inhibitor trichostatin A (TSA). To examine the contribution of MEIS proteins to this response, we used the chromatin immunoprecipitation assay to show that MEIS1 in addition to PBX1, HOXA1, and HOXB1 was recruited to a known PBX.HOX target, the Hoxb1 autoregulatory element following Hoxb1 transcriptional activation in P19 cells. Subsequent to TSA treatment, MEIS1 recruitment lagged behind that of HOX and PBX partners. MEIS1A also enhanced the transcriptional activation of a reporter construct bearing the Hoxb1 autoregulatory element after treatment with TSA. The MEIS1 homeodomain and protein-protein interaction with PBX contributed to this activity. We further mapped TSA-responsive and CREB-binding protein-dependent PKA-responsive transactivation domains to the MEIS1A and MEIS1B C termini. Fine mutation of the 56-residue MEIS1A C terminus revealed four discrete regions required for transcriptional activation function. All of the mutations impairing the response to TSA likewise reduced activation by PKA, implying a common mechanistic basis. C-terminal deletion of MEIS1 impaired transactivation without disrupting DNA binding or complex formation with HOX and PBX. Despite sequence similarity to MEIS and a shared ability to form heteromeric complexes with PBX and HOX partners, the PREP1 C terminus does not respond to TSA or PKA. Thus, MEIS C termini possess transcriptional regulatory domains that respond to cell signaling and confer functional differences between MEIS and PREP proteins. PMID: 15654074 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Blood. 2005 Feb 15;105(4):1456-66. Epub 2004 Nov 2. HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo. Fischbach NA, Rozenfeld S, Shen W, Fong S, Chrobak D, Ginzinger D, Kogan SC, Radhakrishnan A, Le Beau MM, Largman C, Lawrence HJ. Department of Medicine, Veterans Affairs Medical Center, San Francisco, CA 94121, USA. fischba@itsa.ucsf.edu The 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. PMID: 15522959 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Clin Endocrinol Metab. 2005 Jan;90(1):522-8. Epub 2004 Oct 19. HOXA10, Pbx2, and Meis1 protein expression in the human endometrium: formation of multimeric complexes on HOXA10 target genes. Sarno JL, Kliman HJ, Taylor HS. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA. HOXA10 is a transcription factor necessary for embryonic uterine development and for adult endometrial receptivity. The three-amino acid loop extension family of cofactors, including Pbx and Meis, provide HOX target gene specificity in development and myeloid differentiation. Here we demonstrate the expression of Pbx and Meis family cofactors in the human endometrium and their interaction with HOXA10. Using immunohistochemical analysis, we found that Pbx2 and Meis1, but not Pbx1, Pbx3, or Meis2, were expressed in human endometrium. HOXA10, Pbx2, and Meis1 were expressed in the stroma throughout the menstrual cycle. The glandular expression of HOXA10 and Meis1 was menstrual cycle stage specific, whereas glandular Pbx2 expression did not vary. Pbx2, but not Meis1, was expressed in Ishikawa cells. EMSA demonstrated HOXA10-Pbx2 binding as a heterodimer to an enhancer of the EMX2 gene, a known target of HOXA10 regulation. Ablation of the Pbx binding site, but not ablation of the HOXA10 binding site in EMX2, resulted in loss of dimer binding. Based on the observed expression and binding patterns of Pbx2, Meis1, and HOXA10, it is likely that heterodimeric and trimeric complexes involving these proteins determine HOXA10 target gene specificity. Enhanced target gene specificity imparted by multimer binding is likely necessary for HOXA10-mediated endometrial receptivity. PMID: 15494461 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Blood. 2005 Feb 1;105(3):1222-30. Epub 2004 Oct 12. Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines. Wermuth PJ, Buchberg AM. Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, Thomas Jefferson University, 233 S 10th St, Philadelphia, PA 19107-5541, USA. Coexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis. PMID: 15479723 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Dev Cell. 2004 Jun;6(6):737-8. PbX marks the spot. Sagerstrom CG. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, LRB822, 364 Plantation Street, Worcester, MA 01605, USA. Pbx and Meis proteins act as cofactors to various transcription factors, but their exact functions have been unclear. A report by Berkes et al. in Molecular Cell now demonstrates that Pbx and Meis may penetrate repressive chromatin to mark specific genes for activation. Publication Types: Review Review, Tutorial PMID: 15177017 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 2004 Jul 16;279(29):30287-97. Epub 2004 May 11. TALE homeodomain proteins regulate gonadotropin-releasing hormone gene expression independently and via interactions with Oct-1. Rave-Harel N, Givens ML, Nelson SB, Duong HA, Coss D, Clark ME, Hall SB, Kamps MP, Mellon PL. Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92903, USA. Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo. PMID: 15138251 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Cell Biochem. 2004 May 1;92(1):147-63. Retinoic acid regulates the expression of PBX1, PBX2, and PBX3 in P19 cells both transcriptionally and post-translationally. Qin P, Haberbusch JM, Soprano KJ, Soprano DR. Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. Pre-B cell leukemia transcription factors (PBXs) are important co-factors for the transcriptional regulation mediated by a number of Hox proteins during embryonic development. It was previously shown that the expression of several Pbx genes is elevated in mouse embryo limb buds and embryonal carcinoma P19 cells upon retinoic acid (RA) treatment although the mechanism of this induction is not well understood. In this report, we demonstrate that PBX1a, PBX1b, PBX2, and PBX3 mRNAs and PBX1/2/3 proteins are induced during endodermal and neuronal differentiation of P19 cells in a RAR-dependent subtype-unspecific manner following RA treatment. The increases in both PBX1 mRNA and PBX3 mRNA levels are secondary responses to RA treatment requiring new proteins synthesis while the increase in PBX2 mRNA is a primary response. The RA-dependent increases in PBX1 mRNA, PBX2 mRNA, and PBX3 mRNA levels are likely to be transcriptionally regulated since the stability of these mRNAs does not change. In addition, the half-lives of PBX1/2/3 proteins are significantly extended by RA treatment. Two possible mechanisms could contribute to the stabilization of PBX proteins: PBX proteins associate with RA-dependent increased levels of MEIS proteins, and RA may decrease the proteasome dependent degradation of PBX proteins. Copyright 2004 Wiley-Liss, Inc. PMID: 15095411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cancer Biol Ther. 2003 Sep-Oct;2(5):518-23. Comment in: Cancer Biol Ther. 2003 Sep-Oct;2(5):524-5. Altered HOX gene expression in human skin and breast cancer cells. Svingen T, Tonissen KF. School of Biomolecular and Biomedical Science, Griffith University, Nathan, Queensland, Australia. Human HOX genes are expressed in a spatio-temporal fashion during embryogenesis and early development where they act as master transcriptional regulators. HOX genes are also expressed in normal adult cells, potentially in a tissue specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, mis-expression of many HOX genes have been shown, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time RT-PCR assays, the expression of 20 HOX genes and two known HOX co-factors, PBX1B and MEIS1, were analysed in human melanoma and breast cancer cell lines, comparing results against non-malignant cells. Alterations in HOX gene expression were observed for all malignant cells examined, with some dysregulated transcript levels seemingly random, and the expression of other HOX genes apparently following the same patterns in both skin and breast cancer establishment. Furthermore, HOX gene expression was correlated with the invasive capacity of the cells. The expression of the HOX co-factors PBX1B and MEIS1 showed no marked changes from the non-malignant to the malignant phenotypes, further indicating that it is dysregulated HOX gene expression, rather than dysregulated gene expression of HOX co-factors, that potentially commit the cell to re-differentiate and undergo oncogenic transformation. PMID: 14614318 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Blood. 2003 Jun 15;101(12):4748-56. Epub 2003 Feb 27. Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene. Okada Y, Nagai R, Sato T, Matsuura E, Minami T, Morita I, Doi T. Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Japan. Platelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously reported that GATA-1 and ETS-1 regulate the rat PF4 promoter and transactivate the PF4 gene. For the present study, we investigated the regulatory elements and their transcription factors responsible for the lineage-specific expression of the PF4 gene. The promoter activities of deletion constructs were evaluated, and a novel regulatory element termed TME (tandem repeat of MEIS1 binding element) (-219 to -182) was defined. Binding proteins to TME were strongly detected in HEL nuclear extracts by electrophoresis mobility shift assay (EMSA), and they were purified by DNA affinity chromatography. By performing Western blottings and supershift assays, the binding proteins were identified as homeodomain proteins, MEIS1, PBX1B, and PBX2. These factors are expressed in megakaryocytes differentiated from CD34+ cells in human cord blood. MEIS1 and PBXs bind to the TME as MEIS1/PBX complexes and activate the PF4 promoter. In nonmegakaryocytic HepG2 cells, GATA-1 and ETS-1 activate the PF4 promoter approximately 10-fold. Surprisingly, we found that additional expression of both MEIS1 and PBX2 multiplied this major activation another 2-fold. This activation was not observed when MEIS1 binding sites in the TME were disrupted. Furthermore, inhibition of the binding of endogenous MEIS1/PBX complexes to the TME decreased the promoter activity by almost one half, in megakaryocytic HEL cells. Thus, these studies demonstrate that the homeodomain proteins, MEIS1, PBX1B, and PBX2, play an important role in megakaryocytic gene expression. PMID: 12609849 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Teratology. 2002 Nov;66(5):224-34. Comment in: Teratology. 2002 Nov;66(5):217-23. PBX, MEIS, and IGF-I are potential mediators of retinoic acid-induced proximodistal limb reduction defects. Qin P, Cimildoro R, Kochhar DM, Soprano KJ, Soprano DR. Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19122, USA. BACKGROUND: Phocomelia, which is primarily due to a disruption in the proximodistal axis, is found in virtually all mouse embryos exposed to high doses of retinoic acid (RA) on 11 days post coitum (dpc). METHODS: To identify genes that potentially mediate the effects of retinoic acid (RA) on limb development, we have examined the expression of 9,000 clones from the IMAGE consortium by microarray analysis of RNA isolated from 11 dpc mouse forelimbs exposed to RA or vehicle for 6 hr. Eight genes that demonstrated altered expression were chosen for further study of their mRNA levels using RT-PCR. Protein levels were determined by Western blot analysis. RESULTS: Of the 9,000 genes examined in the microarray, approximately 111 demonstrated altered expression (33 known genes and 78 ESTs). Of the eight known genes chosen for further study using RT-PCR, four mRNAs (PBX1a, PBX1b, IGF-Ia, and IGF-Ib) demonstrated consistent elevation ( approximately 3-fold) in their levels after RA treatment in both the forelimbs and hindlimbs as early as 3 hr after RA treatment. In addition to the two PBX1 isoforms, the mRNA level of the other two subtypes (PBX2 and PBX3) and the level of PBX1/2/3 protein were also found to be elevated in limb buds after RA treatment. Finally, we examined the expression of MEIS1, MEIS2, and MEIS3 because these proteins are necessary for PBX nuclear localization. The mRNA level of all three subtypes of MEIS were elevated approximately three- to four-fold in both the forelimbs and hindlimbs after RA treatment. CONCLUSIONS: Because both PBX and MEIS (and their orthologs) are believed to be involved in the control of proximodistal axis formation in mouse and fly limbs and IGFs in the development of limbs, we suggest that increases in PBX, MEIS and IGF-1 mRNA levels may contribute to proximodistal limb reduction defects caused by teratogenic doses of RA. Copyright 2002 Wiley-Liss, Inc. PMID: 12397630 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5448-53. Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos. Maeda R, Ishimura A, Mood K, Park EK, Buchberg AM, Daar IO. Regulation of Cell Growth Laboratory, National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD 21702, USA. Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development. PMID: 11960001 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Exp Hematol. 2002 Jan;30(1):49-57. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada. OBJECTIVE: The Hox gene family of transcription factors is thought to be involved in the regulation of primitive hematopoietic cells, including stem cells and early committed progenitors, and has also been directly implicated in leukemia. To gain further insight into Hox gene-mediated regulation of hematopoiesis, we investigated the expression pattern of representative Hox genes and two of their cofactors, Pbx1 and Meis1, at different stages of murine hematopoiesis. METHODS: Functionally distinct subpopulations of murine bone marrow (BM) and fetal liver day 14.5 (FL) cells were isolated by flow cytometry, and gene expression of various homeobox-containing genes was assessed by global cDNA amplification technique. RESULTS: Hox genes were found preferentially expressed in hematopoietic stem cell (HSC)-enriched subpopulations and downregulated following differentiation and maturation. This profile of expression was observed at both adult and fetal stages of hematopoiesis. The Pbx1 and Meis1 genes had important differences in their expression pattern but were both detected in Hox expressing subpopulations. In particular, Meis1 consistently showed an expression profile closely resembling that of Hox genes. Finally, using the in vitro embryonic stem (ES) cell differentiation model to mimic embryonic hematopoiesis, we found coexpression of Hox genes and their cofactors coincided with the appearance of hematopoietic progenitor cells. CONCLUSION: Together, these results further support the notion that Hox genes are involved in the regulation of early hematopoietic cells and provide strong evidence that they are involved in the regulation of hematopoiesis throughout ontogeny. PMID: 11823037 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Biol Chem. 2001 Jun 29;276(26):23838-48. Epub 2001 Apr 30. The homeodomain proteins PBX and MEIS1 are accessory factors that enhance thyroid hormone regulation of the malic enzyme gene in hepatocytes. Wang Y, Yin L, Hillgartner FB. Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, West Virginia 26506, USA. Triiodothyronine (T3) stimulates a robust increase (>40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes. Previous work has shown that optimal T3 regulation of malic enzyme transcription is dependent on the presence of an accessory element (designated as region E) that immediately flanks a cluster of five T3 response elements in the malic enzyme gene. Here, we have analyzed the binding of nuclear proteins to region E and investigated the mechanism by which region E enhances T3 responsiveness. In nuclear extracts from hepatocytes, region E binds heterodimeric complexes consisting of the homeodomain proteins PBX and MEIS1. Region E contains four consecutive PBX/MEIS1 half-sites. PBX-MEIS1 heterodimers bind the first and second half-sites, the third and fourth half-sites, and the first and fourth half-sites. The configuration conferring the greatest increase in T3 responsiveness consists of the first and fourth half-sites that are separated by 7 nucleotides. Stimulation of T3 response element functions by region E does not require the presence of additional malic enzyme sequences. In pull-down experiments, PBX1a and PBX1b specifically bind the nuclear T3 receptor-alpha, and this interaction is enhanced by the presence of T3. A T3 receptor-alpha region containing the DNA binding domain plus flanking sequences (amino acids 21-157) is necessary and sufficient for binding to PBX1a and PBX1b. These results indicate that PBX-MEIS1 complexes interact with nuclear T3 receptors to enhance T3 regulation of malic enzyme transcription in hepatocytes. PMID: 11331288 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Mol Cell Biol. 2001 Jan;21(1):224-34. Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia. Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G. Laboratory of Molecular Genetics of Hemopoietic Stem Cells, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada. Complex genetic and biochemical interactions between HOX proteins and members of the TALE (i.e., PBX and MEIS) family have been identified in embryonic development, and some of these interactions also appear to be important for leukemic transformation. We have previously shown that HOXA9 collaborates with MEIS1 in the induction of acute myeloid leukemia (AML). In this report, we demonstrate that HOXB3, which is highly divergent from HOXA9, also genetically interacts with MEIS1, but not with PBX1, in generating AML. In addition, we show that the HOXA9 and HOXB3 genes play key roles in establishing all the main characteristics of the leukemias, while MEIS1 functions only to accelerate the onset of the leukemic transformation. Contrasting the reported functional similarities between PREP1 and MEIS1, such as PBX nuclear retention, we also show that PREP1 overexpression is incapable of accelerating the HOXA9-induced AML, suggesting that MEIS1 function in transformation must entail more than PBX nuclear localization. Collectively, these data demonstrate that MEIS1 is a common leukemic collaborator with two structurally and functionally divergent HOX genes and that, in this collaboration, the HOX gene defines the identity of the leukemia. PMID: 11113197 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Genes Dev. 2000 Oct 15;14(20):2581-6. p21 is a transcriptional target of HOXA10 in differentiating myelomonocytic cells. Bromleigh VC, Freedman LP. Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. The myeolomonocytic cell line U937 differentiates into macrophages in response to a variety of agents. Several genes including the cyclin-dependent kinase inhibitor p21(waf1/cip1) and the homeobox gene transcription factor HOXA10 are induced at the onset of differentiation. Ectopic expression of either gene results in U937 differentiation. In this paper, we describe a mechanism by which p21 and HOXA10 may act in concert, where HOXA10 can bind directly to the p21 promoter and, together with its trimeric partners PBX1 and MEIS1, activate p21 transcription, resulting in cell cycle arrest and differentiation. These experiments for the first time identify p21 as a selective target for a HOX protein and link the differentiative properties of a transcription factor and a cell cycle inhibitor. PMID: 11040212 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Exp Cell Res. 2000 Oct 10;260(1):105-15. A conformational change in PBX1A is necessary for its nuclear localization. Saleh M, Huang H, Green NC, Featherstone MS. McGill Cancer Centre, McGill University, Montreal, Quebec, H3G 1Y6, Canada. The fly homeodomain (HD) protein EXTRADENTICLE (EXD) is dependent on a second HD protein, HOMOTHORAX (HTH), for nuclear localization. We show here that in insect cells the mammalian homolog of EXD, PBX1A, shows a similar dependence on the HTH homologs MEIS1, 2, and 3 and the MEIS-like protein PREP1. Paradoxically, removal of residues N-terminal to the PBX1A HD abolishes interactions with MEIS/PREP but allows nuclear accumulation of PBX1A. We use deletion mapping and fusion to green fluorescent protein to map two cooperative nuclear localization signals (NLSs) in the PBX HD. The results of DNA-binding assays and pull-down experiments are consistent with a model whereby the PBX N-terminus binds to the HD and masks the two NLSs. In support of the model, a mutation in the PBX HD that disrupts contact with the N-terminus leads to constitutive nuclear localization. The HD mutation also increases sensitivity to protease digestion, consistent with a change in conformation. We propose that MEIS family proteins induce a conformational change in PBX that unmasks the NLS, leading to nuclear localization and increased DNA-binding activity. Consistent with this, PBX1 is nuclear only where Meis1 is expressed in the mouse limb bud. Copyright 2000 Academic Press. PMID: 11010815 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Mech Dev. 2000 Jun;94(1-2):183-7. Expression of Meis and Pbx genes and their protein products in the developing telencephalon: implications for regional differentiation. Toresson H, Parmar M, Campbell K. Wallenberg Neuroscience Center, Division of Neurobiology, Section for Developmental Neurobiology, Lund University, Sweden. hakan.toresson@mphy.lu.se The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423-429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171-183; Kurrant et al., 1998. Development 125, 1037-1048; Pai et al., 1998. Genes Dev. 12, 435-446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon. PMID: 10842069 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Oncogene. 1999 Dec 23;18(56):8033-43. An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1. Calvo KR, Knoepfler P, McGrath S, Kamps MP. Department of Pathology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, California, CA 92093, USA. The Pbx/Exd family of homeodomain (HD) proteins contribute to the transcriptional and developmental roles of other Hox and Meis/Prep1/Hth HD proteins through heterodimer formation. E2a-Pbx1 is an oncogenic derrivative of Pbx1 produced by the t(1;19) translocation in pediatric pre-B cell acute lymphoblastic leukemia. E2a-Pbx1 heterodimerizes with Hox but not with Meis/Prep1 proteins, produces acute myeloid leukemia in mice, and blocks differentiation of cultured murine myeloid progenitors. Here, we characterize negative and positive regulatory sequences that flank the Pbx1 HD and determine their importance for myeloid immortalization by E2a-Pbx1. A 25 residue predicted alpha helix preceding the Pbx1 HD bound the HD and prevented both its binding to DNA and its ability to heterodimerize with Hox proteins. Addition of 39 residues N-terminal to this inhibitory helix exposed a Pbx dimerization interface that orchestrated cooperative DNA-binding of E2a-Pbx1 and all Pbx proteins as homodimers and heterdimers. Sequences inhibiting DNA-binding and mediating Pbx dimerization coincided with those reported to have nuclear export function. An additional 103 residues N-terminal to the Pbx dimerization interface restored heterodimerization with Hox and Meis1/Prep1 proteins. This negative switch domain - comprised of the inhibitory helix and N-terminal regions required for its partner-mediated derepression - was dispensable for myeloid immortalization by E2a-Pbx1. While stabilizing the heterodimer, the 310 helix C-terminal to the Pbx1 HD was also dispensable for the ability of E2a-Pbx1 to heterodimerize with Hox proteins and immortalize myeloblasts. Retention of myeloid immortalization by E2a-Pbx1 proteins lacking all Pbx1 sequences N- or C-terminal to the HD indicates that Hox proteins, or a yet undefined factor that binds the Pbx1 HD and derepresses DNA-binding by the HD, cooperate with E2a-Pbx1 in myeloid immortalization. PMID: 10637514 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Nature. 1999 Nov 25;402(6760):425-9. Conserved regulation of proximodistal limb axis development by Meis1/Hth. Mercader N, Leonardo E, Azpiazu N, Serrano A, Morata G, Martinez C, Torres M. Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia, CSIC-UAM, Madrid, Spain. Vertebrate limbs grow out from the flanks of embryos, with their main axis extending proximodistally from the trunk. Distinct limb domains, each with specific traits, are generated in a proximal-to-distal sequence during development. Diffusible factors expressed from signalling centres promote the outgrowth of limbs and specify their dorsoventral and anteroposterior axes. However, the molecular mechanism by which limb cells acquire their proximodistal (P-D) identity is unknown. Here we describe the role of the homeobox genes Meis1/2 and Pbx1 in the development of mouse, chicken and Drosophila limbs. We find that Meis1/2 expression is restricted to a proximal domain, coincident with the previously reported domain in which Pbx1 is localized to the nucleus, and resembling the distribution of the Drosophila homologues homothorax (hth) and extradenticle (exd); that Meis1 regulates Pbx1 activity by promoting nuclear import of the Pbx1 protein; and that ectopic expression of Meis1 in chicken and hth in Drosophila disrupts distal limb development and induces distal-to-proximal transformations. We suggest that restriction of Meis1/Hth to proximal regions of the vertebrate and insect limb is essential to specify cell fates and differentiation patterns along the P-D axis of the limb. PMID: 10586884 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Mol Cell Biol. 1999 Nov;19(11):7577-88. PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins. Shanmugam K, Green NC, Rambaldi I, Saragovi HU, Featherstone MS. McGill Cancer Centre, McGill University, Montreal, Quebec, Canada H3G 1Y6. HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively. PMID: 10523646 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Nucleic Acids Res. 1999 Sep 15;27(18):3752-61. A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1. Knoepfler PS, Bergstrom DA, Uetsuki T, Dac-Korytko I, Sun YH, Wright WE, Tapscott SJ, Kamps MP. Department of Basic Science, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription. PMID: 10471746 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Mol Cell Biol. 1999 Jul;19(7):5134-42. Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity. Jacobs Y, Schnabel CA, Cleary ML. Department of Pathology, Stanford University Medical Center, Stanford, California 94305, USA. Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. PMID: 10373562 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Endocr Res. 1998 Aug-Nov;24(3-4):489-95. The bovine CYP17 promoter contains a transcriptional regulatory element cooperatively bound by tale homeodomain proteins. Bischof LJ, Kagawa N, Waterman MR. Department of Biochemistry, Vanderbilt University, Nashville, TN, USA. Bovine CYP17 is regulated at the transcriptional level by ACTH acting through the second messenger cAMP in adrenal fasciculata and reticularis cells. Promoter analysis has previously identified two regions, proximal and distal, within the CYP17 promoter important in the cAMP dependent transcriptional regulation of this gene. The proximal (-80 to -40) cAMP responsive sequence (CRS2) has been identified as a binding site for Steroidogenic Factor-1 (SF-1)/Ad4BP. The distal region (-243 to -100) is also important for the cAMP transcriptional response as revealed by deletion analysis. Within this distal region from -243 to -225, an independent cAMP responsive sequence referred to as CRS1 has been described. The transcription factors binding CRS1 have been identified as homeodomain transcription factors belonging to an atypical class of homeodomain proteins referred to as TALE. Two families of homeodomain proteins which bind CRS1 are the Pbx and Meis1 families. Proteins from neither of these families can bind CRS1 individually; however, members of the Pbx family interact with members of the Meis1 family to cooperatively bind this element. CRS1 was the first identified cis-acting target element for members of both the Pbx and Meis1 family. Unlike SF-1, these proteins are not expressed in a steroidogenic tissue-specific manner but rather, appear ubiquitous. A current model for the function of these proteins in CYP17 regulation is that they may enhance the cAMP response through the downstream SF-1 binding site. PMID: 9888529 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: EMBO J. 1998 Jul 1;17(13):3714-25. Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Laboratory of Molecular Genetics of Hemopoietic Stem Cells, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7. Hoxa9, Meis1 and Pbx1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either Pbx1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor-dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b failed to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells. PMID: 9649441 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Biol Chem. 1998 Apr 3;273(14):7941-8. Members of the meis1 and pbx homeodomain protein families cooperatively bind a cAMP-responsive sequence (CRS1) from bovine CYP17. Bischof LJ, Kagawa N, Moskow JJ, Takahashi Y, Iwamatsu A, Buchberg AM, Waterman MR. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. Bischolj@ctrvax.vanderbilt.edu The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein upon purification using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs extradenticle from Drosophila melanogaster and ceh-20 from Caenorhabditis elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permit these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence. PMID: 9525891 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: EMBO J. 1998 Mar 2;17(5):1423-33. Prep1, a novel functional partner of Pbx proteins. Berthelsen J, Zappavigna V, Mavilio F, Blasi F. Dipartimento di Genetica e Biologia dei Microrganismi dell'Universita, DIBIT, H.S. Raffaele, via Olgettina 58, 20132, Milan, Italy. The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo. PMID: 9482739 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14553-8. Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1. Knoepfler PS, Calvo KR, Chen H, Antonarakis SE, Kamps MP. Department of Pathology, University of California at San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA. pknoepfler@ucsd.edu E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx-Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1-HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx-Hox complexes. In addition, because most motifs bound by Pbx-Meis1/pKnox1 were not bound by Pbx1-Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx-Hox motifs to those containing Pbx-Meis motifs. PMID: 9405651 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Mol Cell Biol. 1997 Nov;17(11):6448-58. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins. Shen WF, Montgomery JC, Rozenfeld S, Moskow JJ, Lawrence HJ, Buchberg AM, Largman C. Department of Medicine, University of California VA Medical Center, San Francisco 94121, USA. Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID: 9343407 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Mol Cell Biol. 1997 Oct;17(10):5679-87. Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins. Chang CP, Jacobs Y, Nakamura T, Jenkins NA, Copeland NG, Cleary ML. Department of Pathology, Stanford University Medical Center, California 94305, USA. The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1. PMID: 9315626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Nat Genet. 1996 Feb;12(2):149-53. Comment in: Nat Genet. 1996 Feb;12(2):113-4. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. Nakamura T, Largaespada DA, Shaughnessy JD Jr, Jenkins NA, Copeland NG. Mammalian Genetics Laboratory, NCI-Frederick Cancer Research and Development Center, Maryland 21702, USA. Retroviruses induce myeloid leukaemia in BXH-2 mice by the insertional mutation of cellular proto-oncogenes or tumour suppressor genes. Disease genes can thus be identified by proviral tagging through the identification of common viral integration sites in BXH-2 leukaemia. Here, we describe a new approach for proviral tagging that greatly facilitates the identification of BXH-2 leukaemia genes. Using this approach, we identify three genes whose expression is activated by proviral integration in BXH-2 leukaemias; Hoxa7, Hoxa9, and a Pbx1-related homeobox gene, Meis1. Proviral activation of Hoxa7 or Hoxa9 is strongly correlated with proviral activation of Meis1 implying that Hoxa7 and Hoxa9 cooperate with Meis1 in leukaemia formation. These studies provide the first genetic evidence that Pbx1-related genes cooperate with Hox genes in leukaemia formation and identify a number of new murine myeloid leukaemia genes. PMID: 8563752 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Mol Cell Biol. 1995 Oct;15(10):5434-43. Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice. Moskow JJ, Bullrich F, Huebner K, Daar IO, Buchberg AM. Jefferson Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA. Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice. PMID: 7565694 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------