PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract

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.     

HOXA9 regulates miR-155 in hematopoietic cells

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9^−/−bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples, as well as in wild-type versus Hoxa9-deficient marrow, using northern analysis and qRT–PCR. Infection of wild-type BM with HOXA9 expressing or GFP⁺ control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors, being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells, indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1, an important regulator of myelopoiesis, was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein, HOXA9 did not appear to modulate Pu.1 expression in murine BM cells.     

Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia

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.     

GATA-1 forms distinct activating and repressive complexes in erythroid cells

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.     

Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells

The structural and functional organization of the adaptor protein Ruk₁ is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk₁ structure involved in protein–protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk₁ with a Glu-epitope at the C-terminus (Ruk₁ Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk₁ Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk₁ Glu-tagged preparation by retardation of electrophoretic mobility of ³²P-labeled oligodeoxyribonucleotides in gel. The Ruk₁ Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl₂ and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk₁ protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80 and 72 kD were obtained. The reaction was inhibited by ZnCl₂ and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk₁ protein was identified in the fractions. It was concluded that the adaptor protein Ruk₁ forms complexes with endonucleases in HEK293 cells.     

DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells

The innate immune system is equipped with many molecular sensors for microbial DNA/RNA to quickly mount antimicrobial host immune responses. In this paper, we identified DHX9, a DExDc helicase family member, as an important viral dsRNA sensor in myeloid dendritic cells (mDCs). Knockdown of DHX9 expression by small heteroduplex RNA dramatically blocked the ability of mDCs to produce IFN-α/β and proinflammatory cytokines in response to polyinosine-polycytidylic acid, influenza A, and reovirus. DHX9 could specifically bind polyinosine-polycytidylic acid via its double-strand RNA binding motifs. DHX9 interacted with IPS-1 via the HelicC-HA2-DUF and CARD domains of DHX9 and IPS-1, respectively. Knockdown of DHX9 expression in mDCs blocked the activation of NF-κB and IFN regulatory factor 3 by dsRNA. Collectively, these results suggest that DHX9 is an important RNA sensor that is dependent on IPS-1 to sense pathogenic RNA.     

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.