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The GATA-1–interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1–independent activation by GATA-1 has been described, all known examples of GATA-1–mediated repression are FOG-1 dependent. In the GATA-1–null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1–binding defective mutant of GATA-1 repressed several genes similarly upon activation with β-estradiol. Repression also occurred in a FOG-1–null cell line expressing ER–GATA-1 and during ex vivo erythropoiesis. At the Lyl1 and Rgs18 loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1–mediated transcriptional regulation.

The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. Previously, we described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia (MDS)/leukemia. In contrast to Gata2 knockout mice, which die around E10.5 because of anemia, the prolonged embryonic development of +9.5 site knockout mice provides a unique model system to investigate the potential roles for GATA-2 in HSC production, migration and function, and more specifically, the requirement for the +9.5 element to regulate Gata2 expression during these processes. Using an ex vivo system involving culturing intact AGM, or AGM dissociated into single cells and then reaggregated into an organoid, we demonstrated that the +9.5 deletion reduced Gata2 expression in uncultured AGM (1.4 fold, p Disclosures: No relevant conflicts of interest to declare.

Abstract 1226 How transcriptional and post-transcriptional mechanisms control the levels/activities of master developmental regulators has fundamental importance for understanding complex developmental processes such as hematopoiesis and associated pathological disorders. GATA-2 is an essential regulator of hematopoiesis, and GATA-2 mutations characterize heritable disease associated with myelodysplastic syndrome and acute myeloid leukemia, including MonoMAC (syndrome of monocytopendia, B and NK cell lymphopenia, and mycobacterial, fungal and viral infection). However, many questions remain unanswered regarding mechanisms underlying GATA-2 regulation and function. We demonstrated that a MonoMAC patient harbors a 28 bp deletion within GATA2 intron 5 that eliminates a conserved E-box and 5 base pairs of an 8 base pair spacer between the E-box and a conserved GATA motif, which constitutes an E-box-GATA composite element. This composite element resides within the +9.5 kb “GATA switch site” that binds GATA-2 and GATA-1 in the transcriptionally active and repressed states, respectively, and confers hematopoietic and vascular endothelial enhancer activities in transgenic mouse embryos. Importantly, this patient lacked mutations in the GATA2 coding sequence characteristic of other MonoMAC patients, but exhibited prototypical MonoMAC. To elucidate the mechanism underlying the function of the +9.5 composite element, we generated a targeted deletion of the murine element, which yielded embryonic lethality at E13 to E14. Prior to death, +9.5−/− mice exhibit reduced liver size, hemorrhaging, and edema. Nucleated primitive red cells are abundant in the +9.5−/− embryos, in contrast to Gata2 knockout mice, which die at approximately E10.5 from anemia due to failure of primitive and definitive hematopoiesis. Furthermore, primitive erythroid (EryP) colony assays conducted with yolk sacs revealed that the mutation does not affect primitive erythroid precursor functionality. However, the +9.5 deletion strongly reduced Gata2 expression at sites of definitive hematopoiesis, including the fetal liver (8.1 fold, P < 0.004) and cultured explants of the hematopoietic stem cell-generating Aortic Gonadal Mesonephric (AGM) region (4.0 fold, P < 0.001). The homozygous mutant animals exhibited a nearly complete loss of hematopoietic stem cells as determined by flow cytometry (20-fold reduction of Lin-Mac1+CD41-CD48-CD150+Sca+Kit+ cells, P < 0.005) and competitive repopulation (complete loss, P < 0.02) assays, as well as progenitors as determined by colony assays (BFU-E, 60-fold reduction, P < 0.002; CFU-GM, 8.8-fold reduction, P < 0.0001; CFU-GEMM, 19-fold reduction, P < 0.001). To investigate the underlying mechanisms, we developed an allele-specific Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay with heterozygous fetal liver cells to test whether the deletion influences Gata2 chromatin accessibility at the +9.5 region. The deletion significantly reduced (8.4 fold reduction, P < 0.001) chromatin accessibility at this region within the mutant allele, while the wild type allele was unaffected. Thus, any potential remaining cis-elements are insufficient to confer chromatin accessibility, supporting a model in which the transcription factors that normally occupy this GATA switch site lose the capacity to access their respective cis-elements in the context of the mutant allele. Our human and murine studies have therefore revealed a cis-element indispensable for the regulation of Gata2 expression in multiple developmental contexts and necessary for the generation of the definitive hematopoietic stem/progenitor cell compartment. As additional elements are likely to confer Gata2 expression in distinct contexts, including primitive erythropoiesis, we have implemented a multi-faceted effort to identify such elements and to compare their mechanisms with that of the +9.5 site, which will provide fundamental insights into genetic mechanisms controlling normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1304 The expression and activities of master regulatory proteins are exquisitely controlled to ensure the proper development of complex organisms. As a master regulator can orchestrate multiple developmental processes, presumably mechanisms involving a large ensemble of cis-elements and trans-acting factors are deployed to establish this control. GATA-2, a master regulator of hematopoiesis expressed in hematopoietic, endothelial, neuronal, and fat cells is likely subject to such complex regulation. GATA-2 occupies dispersed sites of the Gata2 locus, highlighting a positive autoregulatory mechanism. As GATA-1 levels rise during erythropoiesis, GATA-1 displaces GATA-2 from the Gata2 locus, instigating repression. “GATA switches” occur at five dispersed Gata2 sites (−77, −3.9, −2.8, −1.8 and +9.5 kb). Targeted deletion of GATA factor switch sites −2.8 and −1.8 revealed that these sites individually contribute to maximal Gata2 expression, but are otherwise dispensable for embryogenesis and steady-state hematopoiesis. Herein, we describe the targeted deletion of an intronic GATA switch site (+9.5) comprised of an E-box GATA factor composite cis-element. Strikingly, whereas the Gata2 gene knockout results in lethal anemia by E10.5, the +9.5 mutation yielded embryonic lethality around E13.5. The +9.5 mutation greatly reduced Gata2 expression in fetal livers, heart and endothelial cells, but not in the brain. Consequently, by E12.5 mutant mice exhibited severely reduced numbers of hematopoietic stem/progenitor cells (HSPCs) in fetal livers as assayed by cell sorting and colony assays. However, mutant mice were not overtly anemic and Ter119+ cells were abundant in the mutant fetal livers, accounting for the delay in lethality compared to the Gata2 knockout. Additionally, +9.5 mutant embryos displayed vascular/cardiovascular malformations and severe hemorrhaging. This work reveals that, unlike the −2.8 and −1.8 GATA switch sites, the +9.5 composite element functions as a “master regulatory cis-element” required for HSPC development, vascular integrity, and cardiovascular development. Genomic analyses identified a large cohort of loci containing conserved intronic composite elements resembling the +9.5 element, with heavy representation at genes critical for HSPC and vascular development/function, as well as novel genes of unknown function. Disclosures: No relevant conflicts of interest to declare.

Key steps in hematopoiesis and the expression of genes encoding hemoglobin subunits are critically dependent upon specific members of the GATA factor family of transcription factors. Our recent efforts have focused on elucidating how GATA factors select functional sites in chromatin and how they function combinatorially with additional regulatory factors. GATA motifs are often arranged in close proximity to E-boxes, and such composite elements commonly mediate GATA factor- and Scl/TAL1-dependent transcriptional responses. Only a small fraction of these composite elements in chromatin are occupied by GATA factors and Scl/TAL1, and a specific epigenetic signature distinguishes occupied versus unoccupied elements genome-wide. In the context of hemoglobin synthesis, we are using genetic and molecular approaches to dissect the multistep mechanism underlying the control of β-globin transcription. GATA-1-containing complexes assemble at the β-globin Locus Control Region (LCR) prior to the murine adult βmajor promoter. Though the LCR physically interacts with the βmajor promoter, this interaction is not required for the binding of several trans-acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems. promoter. Though the LCR physically interacts with the β promoter, this interaction is not required for the binding of several -acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems.

Miyakoshi et al[1][1] recently reported on 4 female Japanese patients with multiple myeloma and prior stem cell transplantation who developed severe pulmonary complications after receiving bortezomib therapy. We have seen a similar complication in an African-American man. The patient is a 66-year

The transcription factors GATA-1 and GATA-2 confer both shared and unique activities to regulate hematopoiesis. GATA-2 is essential for the survival and proliferation of hematopoietic stem cells and multipotent hematopoietic precursors, whereas GATA-1 is critical for erythropoiesis. GATA-1 and GATA-2 are expressed reciprocally during erythropoiesis, with GATA-2 expression preceding that of GATA-1. Erythroid maturation appears to require GATA-2 repression. We have shown that chromatin-bound GATA-2 is displaced by GATA-1, which instigates a distinct transcriptional output at the respective locus. We hypothesize that such “GATA switches” are an important transcriptional mechanism that drives erythropoiesis. Their distinct transcriptional outputs suggest that GATA-1 and GATA-2 have unique biochemical properties. However, biochemical analyses have primarily identified similarities between the factors. For instance, the two zinc fingers of GATA-1 are highly homologous to those of GATA-2, and therefore both factors interact with the cofactor FOG-1 and bind an identical WGATAR sequence with high affinity. We found that GATA-2 is considerably less stable than GATA-1. Studies using cycloheximide to block protein synthesis in erythroid precursor cell lines derived from embryonic stem cells indicate that the half-life of endogenous GATA-2 is ~45–90 min, whereas that of endogenous GATA-1 is >4 h. Biosynthetic labeling studies to measure the stabilities of endogenous GATA-1 and GATA-2 expressed in the same cell confirmed that GATA-2 is considerably less stable than GATA-1. GATA-2 levels increase in cells treated with the proteasome inhibitors MG132 or clasto-lactacystin β-lactone. Importantly, we have found that rapid GATA-2 proteolysis is essential for the proper temporal control of erythroid-specific gene transcription. Quantitative chromatin immunoprecipitation analysis revealed that proteasomal inhibition attenuated the ability of GATA-1 to displace GATA-2 from target genes, which are regulated by GATA switches. Proteasomal inhibition resulted in impaired GATA-2 displacement from GATA-2, and consequently a significantly lower degree of GATA-2 repression. Proteasomal inhibition also diminished GATA-1-mediated displacement of GATA-2 from GATA-1, α-globin, and ALAS-2, genes at which GATA switches precede transcriptional activation. We have therefore identified the 26S proteasome as a key regulator of GATA factor exchange on chromatin. Our studies indicate that rapid proteasomal degradation of GATA-2 is required for GATA-1 and GATA-2 to establish unique patterns of gene expression during erythropoiesis. Studies are underway to determine whether GATA-2 is similarly unstable in multipotent hematopoietic precursors, or if its instability is established upon commitment to the erythroid lineage.

A 59 year-old Caucasian male with a history of hepatitis C infection, presented in October 2003 with a painless skin lesion on his back. Physical examination revealed a 3 cm lesion on the back that resembled a sebaceous cyst, with similar lesions on the anterior aspects of both legs. A right inguinal lymph node was palpated as well, measuring approximately 2 cm. A wide surgical resection was performed on the back lesion due to its extensive nature at the time of surgery. Pathologic evaluation revealed a primary cutaneous anaplastic large cell lymphoma, measuring 12 cm x 5 cm in size. Immunohistochemical staining showed reactivity for CD30, CD3, UCHL-1, CD43 and CD45. Despite being asymptomatic, the patient had widespread disease on CT/PET scans, including diffuse lymphadenopathy, bony disease, and lung lesions. The patient was treated with weekly high-dose methotrexate at 2 grams/m2 followed by leucovorin rescue for cycle 1 and dose-reduced for cycles 2 through 5 to 1 gram/m2 secondary to myelosuppression. For cycles 6 through 10, the patient was able to tolerate the original dose of 2 grams/m2. After eight cycles, the patient had complete resolution of all disease by clinical examination and by CT/PET scans, and has remained in complete remission since February 2004. The patient tolerated the chemotherapy well with myelosuppression being the most significant toxicity. He also had a transient increase in his liver enzymes after cycle 1 that resolved spontaneously. He is currently being treated for reactivation of hepatitis C with pegylated interferon and ribavirin. To the best of our knowledge, this is the first case of primary cutaneous anaplastic large cell lymphoma with systemic involvement being treated successfully with weekly high-dose methotrexate and leucovorin rescue with prolonged complete remission. Weekly high-dose methotrexate with leucovorin rescue is an active and well-tolerated regimen with limited side effects.

There is increasing recognition that autoimmune hemolytic anemia (AIHA) may be associated with alloreactivity to red blood cell antigens (Young et al, Transfusion44: 67, 2004; Zumberg et al, Arch Intern Med16: 285, 2001). We report a patient who developed refractory AIHA and alloantibodies to Rh antigens 3 months after a non-myeloablative haploidentical transplant and our attempt to support her with polymerized human hemoglobin transfusions. This 44 year old AB Rh-negative female with del(7) acute leukemia attained a complete remission after standard antileukemic therapy. One month later, she received a haploidentical stem cell transplant from her A Rh-negative sister; preparative drugs included cyclophosphamide, fludarabine, and alemtuzumab. The transplant failed to engraft, confirmed by chimerism studies, and cyclosporine and mycophenolate prophylaxis was discontinued. Treatment for cytomegalovirus (CMV) antigenemia and staphylococcal bacteremia during the next 6 weeks resulted in severe thrombocytopenia for which she received platelet transfusions. Bone marrow biopsy revealed normal chromosomes and no evidence of leukemia, and her infections resolved. Three months after transplant, she developed Evans’ syndrome with severe AIHA and worsening thrombocytopenia. She had a panagglutinin (DAT IgG+, C3+) and was found to have anti-C, D, and E alloantibodies. Previous indirect antibody screens were negative. For 3 weeks, hemolysis continued unabated, requiring 54 units of blood despite treatment with steroids, rituximab, alemtuzumab, plasmapheresis, and erythropoietin. She developed marked hyperbilirubinemia and acute renal failure and required mechanical ventilation and hemodialysis. CMV assays were negative. Due to concerns that transfusions were playing a role in her multiorgan failure, we requested and received permission, despite doubts of its benefit in this setting, for emergency use of the investigational polymerized pyridoxilated stroma-free hemoglobin PolyHemeTM (Northfield Laboratories Inc). She received 11 units over 56 hours raising her hemoglobin to 7.0 g/dl while her hematocrit dropped to

Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, analyses of GATA-1 occupancy by chromatin immunoprecipitation (ChIP) revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the −2.8 kb region of the GATA-2 locus in the active state, despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high-resolution ChIP to show that GATA-1 and GATA-2 occupy two additional regions, −3.9 kb and −1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the −3.9 kb and −1.8 kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and loss of GATA-2 occupancy at the sites. As the distinct preferences for GATA factor occupancy were not evident from electrophoretic mobility shift assay analysis in vitro, establishment of the preferences requires the native nucleoprotein structure of the GATA-2 locus. In the transcriptionally active state, the −3.9 kb and −1.8 kb regions formed strong DNaseI hypersensitive sites (HSs), and the −2.8 kb region formed a weak HS. Whereas ER-GATA-1-instigated repression abolished the −1.8 kb HS, the −3.9 kb HS persisted in the inactive state. Transient transfection analysis provided evidence that the −3.9 kb region functions distinctly from the −2.8 kb and −1.8 kb regions. Molecular studies, including capturing chromosome conformation (3C) analysis, are underway to dissect the structure/function of the upstream regulatory regions. Based on the high sequence conservation and occupancy by GATA factors in vivo, targeted deletions of the −2.8 kb and −1.8 kb regions were generated. Mutant mice are being developed, and functional analysis is being conducted via in vitro differentiation of homozygous mutant ES cells. Our results support a model in which dynamic changes in GATA factor occupancy and chromatin structure at the −3.9 kb, −2.8 kb, and −1.8 kb regions are intimately controlled by Friend of GATA-1 and additional coregulators. This GATA factor interplay is essential for GATA-2 transcriptional regulation and therefore the control of hematopoiesis.