Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.

Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often over-expressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.

Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5'-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Serum response factor (Srf) is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to Srf(F/F) mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/Srf(F/F) knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41(+) megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

OBJECTIVE:Mutations in the CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPepsilon(-/-) (knockout [KO]) mice, the mechanisms by which C/EBPepsilon mutations act to decrease neutrophil function are not entirely clear. MATERIALS AND METHODS:In order to determine the role of C/EBPepsilon in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPepsilon KO and wild-type mice and performed expression and flow cytometric analysis and functional studies. RESULTS:Expression of lineage-specific cell surface antigens on our in vitro differentiated cell lines revealed persistent expression of monocytic markers on KO granulocytes. We verified this in primary murine peripheral blood and bone marrow cells. In addition, KO bone marrow had an increase in immature myeloid precursors at the common myeloid progenitor and granulocyte/monocyte progenitor levels, suggesting a critical role for C/EBPepsilon not only in granulocyte maturation beyond the promyelocyte stage, but also in the monocyte/granulocyte lineage decision. We found that restoration of Hlx (H2.0-like homeo box 1) expression, which was decreased in C/EBPepsilon KO cells, rescued chemotaxis, but not the other defects of C/EBPepsilon KO neutrophils. CONCLUSIONS:We show two new regulatory functions of C/EBPepsilon in myelopoiesis: in the absence of C/EBPepsilon, there is not only incomplete differentiation of granulocytes, but myelopoiesis is disrupted with the appearance of an intermediate cell type with monocyte and granulocyte features, and the neutrophils have abnormal chemotaxis. Restoration of expression of Hlx provides partial recovery of function; it has no effect on neutrophil maturation, but can completely ameliorate the chemotaxis defect in C/EBPepsilon KO cells.

The MDS1-EVI1 locus is of considerable interest due to its role in myeloid malignancies and dysplasias. It is well established now that the locus has two different transcription start sites (TSS) located 0.5 Mb apart, and these have the capacity to encode different isoforms, which variably contain zinc finger DNA binding domains and a SET-like domain that may have histone modifying ability. In order to better understand the biological role of this locus, we knocked in a lacZ allele into the Mds1 (upstream) TSS by homologous recombination in ES cells and created mice harboring this modified allele (K. Lezon-Geyda, S. Lin, G. Steele-Perkins et al, in preparation). By staining for beta-galactosidase activity, we documented the distribution of Mds1 activity during embryonic development and in the adult. During development, five major organ systems showed expression: musculoskeletal, renal, cardiac, neural, and hematopoietic, and in the latter three, there was a striking and highly specific spatiotemporal pattern of expression suggesting that Mds1-Evi1 plays important regulatory roles. In the developing heart, staining was seen in the anterior heart field specifically during the formation of the cardiac outflow tract, with significant spatiotemporal overlap with Mef2c, which encodes an important cardiac transcriptional regulatory protein. Thereafter, expression in the heart is very low. Beta-gal staining in the hematopoietic system in the embryo is limited to the clusters of nascent hematopoietic progenitors that develop at day 9.5 p.c. in the ventrolateral wall of the dorsal aorta and bud into the vascular lumen. Strikingly, we see no staining in other endothelium, nor in the fetal liver of 12.5–14.5 day embryos, wherein the majority of fetal hematopoiesis takes place. In adult bone marrow, there is beta-gal activity exclusively in the lin− c-kit+ Sca1+ progenitor population, with all of the beta-gal-positive cells being in the progenitor pool, and nearly all of the progenitor cells staining. While homozygous mice are viable, they are small, kyphotic, and have a shortened lifespan. Morphologic and quantitative analysis of the peripheral blood failed to reveal any significant abnormality. To assess the function of the hematopoietic system more rigorously, competitive repopulations of homozygous Mds1-deficient marrow progenitors with wildtype progenitors were performed. Within several weeks after transplant, the Mds1−/− cells were undetectable in the recipients, revealing that the homozygous Mds1-null bone marrow progenitors are deficient in their repopulating ability. To identify what function Mds1-Evi1 plays in hematopoetic cells, we used shRNA to suppress its expression in the myeloid cell lines 32Dcl3 and DA-1. This revealed an increase in steady state levels of cell death, as documented by histone release, TUNEL staining, and caspase activation. These data suggest that a primary role for the Mds1-Evi1 locus in hematopoietic cells is to promote their survival, thus allowing normal expansion at the progenitor stage.

Anemia is the most common hematologic morbidity in HIV+ patients and is associated with increased mortality. In previous studies, we have examined anemia-associated mortality in over 30,000 HIV+ men cared for within the VA system, and have confirmed the increased mortality previously shown to be associated with anemia. Most strikingly, however, we have demonstrated that macrocytosis in particular is an independent risk factor for early mortality even after correcting for alcohol abuse, liver disease, or therapy with AZT. We hypothesize that macrocytosis reflects a proinflammatory state associated with HIV and magnified by co-morbidities. We have therefore sought to link anemia with known markers of underlying inflammation. Genetic variants that affect the level of cytokine gene expression have been shown to influence the development of anemia associated with a variety of illnesses. One such polymorphism occurs in the macrophage migration inhibitory factor (MIF) gene, a cytokine secreted by macrophages and T-cells that induces release of TNF and IL-6 from macrophages. Our group has defined two polymorphisms in the promoter of the human MIF gene that affect the level of MIF expression. The first, a tetranucleotide CATT repeat, occurs 5-8 times within a Pit-1transcription factor binding site, and increased numbers of repeats are associated with higher levels of MIF expression. The second, a single-nucleotide polymorphism (SNP), has been identified at −173 bp (G/C), although its significance in predicting cytokine levels has been hard to separate from the associated CATT repeat polymorphism. High expression MIF polymorphisms have been shown to correlate with the development of anemia in the setting of malaria, to influence recovery from community acquired pneumonia, and to increase the severity of manifestations of rheumatoid arthritis. We therefore undertook to examine whether MIF polymorphisms might influence the development of anemia in the setting of HIV. We wished to test the hypothesis that the presence of high-expressing alleles of MIF predisposes to the development of anemia in the setting of HIV, particularly in association with other co-morbid conditions. In order to address this question, genomic DNA isolated/extracted from blood collected as part of the Veterans Aging Cohort Study (VACS) has been genotyped for the polymorphisms using a PCR based method. These data are being correlated with anemia, mean corpuscular volume, and CD4 count. We are using Cox proportional hazards models stratified by age, race, HIV severity, and co-morbid disease to examine associations between anemia type/severity and MIF gene polymorphisms. We are currently analyzing the prevalence of high-expressing MIF alleles in non-anemic and anemic patients with and without HIV, using multivariate analysis to identify correlations of genotype with markers of morbidity. It is hoped that these studies will provide insight into the etiology of anemia in HIV+ patients in general, and of macrocytic anemia in particular.

The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation.

With the goal of creating a resource for in-depth study of myelopoiesis, we have executed a 2-pronged strategy to obtain a complementary DNA (cDNA) clone set enriched in hematopoietic genes. One aspect is a library subtraction to enrich for underrepresented transcripts present at early stages of hematopoiesis. For this, a hematopoietic cDNA library from primary murine bone marrow cells enriched for primitive progenitors was used as tester. The subtraction used 10 000 known genes and expressed sequence tags (ESTs) as driver. The 2304 randomly picked clones from the subtracted cDNA libraries represent 1255 distinct genes, of which 622 (50%) are named genes, 386 (30%) match uncharacterized ESTs, and 247 (20%) are novel. The second aspect of our strategy was to complement this subtracted library with genes known to be involved in myeloid cell differentiation and function. The resulting cDNAs were arrayed on polylysine-coated glass slides. The microarrays were used to analyze gene expression in primary and cultured murine bone marrow-derived progenitors. We found expression of various types of genes, including regulatory cytokines and their receptors, signal transduction genes, and transcription factors. To assess gene expression during myeloid differentiation, we examined patterns of change during induced differentiation of EML cells. Several hundred of the genes underwent fluctuations in expression level during myeloid cell differentiation. The complete database, accessible on the World Wide Web at http://yale130132115135.med.yale.edu/, allows for retrieval of information regarding these genes. Our microarray allows for genomewide expression analysis of myeloid stem cells, which will help in defining the regulatory mechanisms of stem cell differentiation.

1183 The small ubiquitin-like modifier (SUMO) can be covalently attached to a large number of proteins through formation of isopeptide bonds with specific lysine residues of target proteins, is covalently attached to substrate proteins via an isopeptide bond between a C-terminal glycine and a lysine residue in the substrate. A consensus SUMO acceptor site has been identified consisting of the sequence ØKXE (Ø is a large hydrophobic amino acid and K is the site of SUMO conjugation). A large number of proteins with important roles in many cellular processes, including gene expression , chromatin structure, signal transduc-tion, and maintenance of the genome, have been found to be SUMOylated (Müller et al., 2001; Gill, 2004). Nuclear proteins, including transcription factors, are a major group of proteins among SUMOylated proteins. These include p53, c-Jun, Sp3, Elk-1, p300 histone acetyltransferase, HDAC (histone deacetylase), PML, HIPKs, and the GATA family proteins. SUMOylation of these proteins has not been generally associated with increased protein degradation. Rather, SUMO modification regulates protein localization, protein–protein or protein–DNA interactions, and/or biochemical activities. The consequence of SUMOylation on protein function is substrate specific. SUMOylation is a dynamic process that is mediated by activating, conjugating, and ligating enzymes and is readily reversed by a family of SUMO-specific proteases (Li and Hochstrasser, 1999). The enzymatic machinery that adds and removes SUMO is similar to, but distinct from, the well known ubiquitination machinery. The E1-activating and E2-conjugating enzymes involved in SUMOylation are highly related to the ubiquitination system. However, the SUMO E3 ligase that promotes transfer of SUMO from the E2 to specific substrates and article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at