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Molecular genetic studies in a seven-year-old boy and his mother demonstrated a novel 11 bp deletion in the TWIST gene (127del11), causing Saethre-Chotzen syndrome. The mother had rather mild signs of the Saethre-Chotzen syndrome; however, her son presented with marked acrocephalosyndactyly type 3, leading to craniotomy at three years. He also had recurrent infections and laboratory findings comparable with the hyper IgE syndrome, a rare primary immunodeficiency disorder. It is likely that the 11bp deletion caused the Saethre-Chotzen syndrome in the patient and his mother, and another, not yet identified genetic defect, seen in the patient but not in the mother, is responsible for the hyper IgE phenotype. A combination of these two congenital conditions has not been described to date.

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.

Studies in mouse, chicken and Xenopus have shown that Slug is selectively expressed in the dorsal part of the developing neural tube. Ablation and antisense experiments in chicken suggest that Slug may be an important factor during neural tube closure. We therefore investigated the role of Slug as a possible candidate contributing to the aetiology of neural tube defects (NTD) in humans. We characterised the genomic structure of human SLUG including determination of the exon-intron boundaries. The coding sequence of SLUG was screened for mutations in 150 patients with NTD using single strand conformation analysis (SSCA). In one patient, we identified a missense mutation 1548C-->A in exon 2 causing an exchange of a conserved amino acid (D119E) in the Slug subfamily-defining region preceding the first zinc finger. This is the first description of a human mutation in the SLUG gene. In accordance with the findings in model organisms, the SLUG mutation may be causally related to the development of NTD in our patient and could be considered as a predisposing factor.

Introduction and Objectives Myc-interacting zinc finger protein 1 (Miz-1; Zbtb17) is a BTB/POZ (POZ) domain transcription factor expressed in all mammalian tissues. The POZ domain is required for Miz-1 function and favors a stable association with chromatin. As a binding partner of the oncoprotein c-Myc, Miz-1 can modulate the expression of c-Myc target genes (e.g. p21Waf1, p15ink4b). c-Myc is frequently associated with a variety of leukemia and lymphoma. In particular, c-Myc takes part in the t(8;14) chromosomal translocation where it is placed under the control of transcriptional regulatory elements of the immunoglobulin heavy chain (IgH) locus. This chromosomal translocation is a hallmark of patients with Burkitt-type B-cell lymphoma (BL). Since epigenetic deregulation is often associated with tumor development, we wished to determine whether (i) Miz-1 is involved in the development of c-Myc-dependent B-cell lymphoma and whether (ii) Miz-1 influences c-Myc dependent chromatin and gene regulation. Methods We are using the Eµ-Myc mouse model to generate Burkitt type c-Myc dependent lymphomas to study the implication of Miz-1 in a c-Myc driven process of malignant transformation. Mice carrying the Eµ-Myc transgene (c-Myc gene placed under the control of Eµ enhancer of the IgH locus) overexpress c-Myc in lymphoid cells and develop a disease similar to Burkitt's lymphoma. Mice that express a conditional non-functional Miz-1 allele lacking the part coding for the POZ domain in B cells (Mb1-Cre-Miz-1DPOZ/flox mice, hereafter called DPOZ mice) were crossed with Eµ-Myc transgenic mice. Incidence and latency periods of the development of tumors in these animals were compared to Eµ-Myc animals that express a functional Miz-1 protein. Pre-cancerous mice of all genotypes were also investigated to evaluate the influence of Miz-1 during the development of the c-Myc dependant lymphomas. To address the role of Miz-1 in the deregulation of chromatin associated with c-Myc overexpression, chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) was performed on B-lymphoma and pre-tumoral B-cells and compared to normal B-cells. Binding to chromatin of Miz-1, c-Myc as well as histone marks will be assessed. RNA from each cell type will also be submitted to high throughput sequencing (RNA-seq) to correlate the binding of transcription factors with gene expression. Results Eµ-Myc mice that express the non-functional Miz-1 protein (DPOZ) in B-cells develop lymphoma later than Eµ-Myc mice that express a normal, functional Miz-1 (Figure 1). Tumors that grow in Eµ-Myc/DPOZ animals are phenotypically similar, but are smaller compared to those developing in Eµ-Myc mice. Additionally, blood analysis of sick animals reveal a lower amount of Large Unstained Cells (LUC) when Miz-1 is mutated. This suggests that Myc driven lymphoma and leukemia is impaired in the absence of a functional Miz-1 protein. Accordingly, in 40 days-old pre-tumoral Eµ-Myc/DPOZ animals compared to Eµ-Myc mice with wild type Miz-1, less pre-B and/or pro-B cells were observed in lymphoid organs and little or no LUC were present in the blood, suggesting that a c-Myc driven B-cell lymphoma develops with a longer latency period and progresses at a slower rate in the absence of a non-functional Miz-1 protein. Conclusion and ongoing work Our preliminary data indicate that Miz-1 is involved in the development of aggressive c-Myc driven B-cell lymphoma. To test the potency of Eµ-Myc/DPOZ tumor cells to initiate B-cell lymphoma, we will transplant these tumor cells into immune-deficient mice and follow the development of the transplanted cells. Also, to test whether Miz-1 would be a suitable therapeutic target for future leukemia and lymphoma treatment, we will use a conditional model (ROSA-CreER-Miz-1DPOZ/flox) where the POZ domain coding sequences of Miz-1 can be deleted after tamoxifen treatment after tumor initiation in Eµ-Myc animals. To better understand the mechanisms by which Miz-1 influences c-Myc in the process of lymphomagenesis, we are performing high throughput genomic analyses to identify genes that are differentially regulated in Eµ-Myc lymphomas when Miz-1 is functional or deficient. These analyzes should demonstrate whether a c-Myc/Miz-1 complex is required to malignantly transform B-cells or whether c-Myc and Miz-1 act independently in the development of B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

The cytokine-inducible transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A and STAT5B) are important for the proper development of multicellular eukaryotes. Disturbed signaling cascades evoking uncontrolled expression of STAT5 target genes are associated with cancer and immunological failure. Here, we summarize how STAT5 acetylation is integrated into posttranslational modification networks within cells. Moreover, we focus on how inhibitors of deacetylases and tyrosine kinases can correct leukemogenic signaling nodes involving STAT5. Such small molecules can be exploited in the fight against neoplastic diseases and immunological disorders.

Abstract 3217 Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells and is required for normal multi-lineage blood cell development. Mutations affecting the function of Gfi1 are implicated in leukemia, lymphoma, auto-immune disease and neutropenia. Gfi1 deficient mice are severely neutropenic and accumulate an aberrant CD11b+GR1int myeloid population, which was assumed to contain precursors either arrested in granulocytic differentiation or redirected into the monocytic lineage. To further analyze the function of Gfi1 during granulopoiesis, we used bone marrow cells from Gfi1-GFP knock-in mice, which enable to follow Gfi1 expression by simply measuring green fluorescence. By this, we found that the CD11b+GR1lo bone marrow fraction that contains monocytes separates into two populations expressing low or high Gfi1. We FACS-sorted these two subsets (CD11b+GR1loGfi1hi and CD11b+GR1loGfi1lo) and observed that, 99% of CD11b+GR1loGfi1lo cells resembled monocytes, while over 90% of the CD11b+GR1loGfi1hi cells were myelocytes or metamyelocytes. After three days in culture in the presence of GM-CSF, almost all CD11b+GR1loGfi1lo cells developed into adhering macrophages, while 73% of the CD11b+GR1loGfi1hi cells developed into mature neutrophils, suggesting that Gfi1 expression drives myeloid precursors into the granulocytic lineage. We determined the genome-wide expression patterns of these two populations and as a control also of CD11b+GR1+ granulocytes and the aberrant CD11b+GR1int population found in Gfi1−/− mice (termed: CD11b+GR1loGfi1−/−). Unsupervised clustering analysis showed that CD11b+GR1loGfi1−/− cells are similar to CD11b+GR1loGfi1lo monocytes, while CD11b+GR1loGfi1hi cells are more closely related to mature granulocytes (CD11b+GR1hi). Gene-Set enrichment analysis (GSEA) revealed that genes associated with cell cycle progression were up regulated in CD11b+GR1loGfi1hi, while genes controlling the humoral immune response and chemokine receptor signaling activity were downregulated. Promoter analysis of differentially expressed genes showed for CD11b+GR1loGfi1hi an over-representation of binding sites for E2F, a regulator of cell cycle genes, and for CD11b+GR1loGfi1lo cells an over-representation of binding sites for STAT5, a transcription factor required for monocyte development. The comparison of CD11b+GR1loGfi1hi cells with granulocytes also showed a higher expression of cell cycle associated genes and a lower expression of humoral immune response genes. This indicated that CD11b+GR1loGfi1hi cells show features of faster cycling, less differentiated precursor cells compared to monocytes (CD11b+GR1loGfi1lo) and granulocytes (CD11b+GR1hi). It is thus likely that CD11b+GR1loGfi1hi monocyte bone marrow subset contains the precursors committed to granulocytic differentiation. The unsupervised clustering analysis also revealed that CD48, a 40–45 kD cell surface glycoprotein, is inversely correlated to the Gfi1 expression and might enable to differentiate between CD11b+GR1loGfi1hi and CD11b+GR1loGfi1lo cells and to find a definition for precursors committed for granulopoiesis. We found that CD48 can replace Gfi1/GFP as well as CD11b in a flow cytometric analysis and using GR1, CD48 staining only, we were able to clearly separate granulocytic precursors from other monocytic cells and mature granulocytes as shown by Wright-Giemsa staining and in vitro culture of sorted cells in the presence of M-CSF or GM-CSF. GR1loCD48hi cells gave rise to adherent macrophages in the presence of M-CSF but generated monocytes and granulocytes in the presence of GM-CSF, while GR1loCD48lo cells can only develop into granulocytes in the presence of GM-CSF. Thus CD48 allowed the separation of bipotential precursors from other precursors that are fully committed to granulopoiesis. Analysis of Gfi1−/− bone marrow cells using the GR1, CD48 markers clearly showed that the aberrant CD11b+Gr1int population represents regular monocytes that accumulated at the expense of granulocytes. This suggests that Gfi1 regulates cell-fate decision in bipotential granulocytic-monocytic precursors, which can be precisely defined by expression of GR1 and CD48. This allows for the first time a more precise definition of monocytic-, granulocytic and bipotential precursors in mice on a phenotypic basis. Disclosures: Duehrsen: Alexion: Honoraria.

Miz-1 (Myc-interacting zinc finger-1) is a BTB/POZ domain transcription factor that can activate or repress gene transcription. To explore the function of Miz-1, we have generated mice that express a non-functional protein lacking the POZ domain, Miz-1ΔPOZ. These mice show a very small thymus owing to a dramatic reduction of early T cell progenitors and DN4 pre-T cells. DN1-3 cells have higher apoptotic rate indicating that Miz-1 is coordinating survival signals. Miz-1ΔPOZ mice also have a DN3/DN4 transitional block and DN3 cells showed a dramtic up-regulation of the cell cycle regulator p21Waf1. Surprisingly, neither the introduction of a Bcl-2 transgene nor the deletion of p21Waf1 rescued the DN3/DN4 block in Miz-1ΔPOZ mice. The block at the pre-TCR-dependent selection stage rather suggested that Miz-1 might regulate pre-TCR signaling. Although the pre-TCR signaling does not seem to be altered by the expression of a non-functional Miz-1, the intracellular expression of the TCRβ is reduced. Introduction of a rearranged TCR transgene rescued the DN3/DN4 cell numbers and their function in Miz-1ΔPOZ mice. Our data suggest that Miz-1 is required for the transport or the stability of the pre-TCR in order to support the proliferative burst of DN3 cells. Comparative gene expression analysis will determine which are the direct effector genes of Miz-1 and contribute to a better understanding of the critical factors involved in this precise step of T cell development.

Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

Inflammatory responses are complex and comprise multiple mediators including cytokines such as TNF-alpha (TNF-α) and IL-1beta. These cytokines are synthesized and secreted in response to signaling by plasma membrane receptors of the Toll-like receptor (TLR) family. A central downstream element of TLR-dependent signaling is the transcription factor NF-kappaB (NF-κB), which plays a pivotal role in controlling the proper sequence of events during an inflammatory response. In unstimulated cells, NF-κB is bound to inhibitory IkappaB (IκB) proteins and remains sequestered in the cytoplasm. Stimulation of TLRs triggers a signaling cascade that leads to phosphorylation and proteasomal degradation of IκB, resulting in the translocation of NF-κB to the nucleus, where it acts as a transcriptional activator of target genes. To keep the innate immune system under control, the TLR signaling cascade is under a tight control of many positive and negative regulators. We have previously shown that the transcription factor Growth Factor Independence 1 (Gfi1) represents a novel factor limiting the inflammatory immune response including TNF-α. Gfi1-deficient (Gfi1−/−) mice show a very strong systemic response to the TLR4 ligand and endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here, we investigated the molecular mechanism of this exaggerated TNF-α production in the absence of Gfi1. It is known that endotoxin stimulation results in the activation of the transcription factor NF-κB through TLR4, leading to TNF-α production. This activation also resulted in rapid and de novo expression of Gfi1 in the nucleus in a time- and dose-dependent manner. The expression of Gfi1 was not due to feedback regulation from secreted TNF, since TNF-deficient macrophages were also able to upregulate Gfi1 mRNA following LPS stimulation. As expected, LPS stimulation of Gfi1−/− macrophages resulted in significantly higher levels of TNF-α mRNA, and secreted TNF-α cytokine. Strikingly and in contrast to most known negative regulators of TLRs, Gfi1 did not affect the activity or the expression levels of the cytoplasmic components of TLR signaling pathway. Additionally, NF-κB phosphorylation and nuclear translocation post- LPS treatment were intact in both Gfi1−/− and Gfi1+/+ macrophages. Immunoprecipitation analysis from cells endogenously expressing Gfi1 and NF-κB or over-expressing these two proteins post transfection, clearly revealed a direct interaction between Gfi1 and the p65 subunit of NF-κB. Immunofluorescence staining of macrophages post-LPS treatment confirmed direct interaction of these two proteins in the nucleus at the endogenous level. Gfi1 represses transcription by binding to DNA recognition sequences in target gene promoters. Thus, aiming to investigate the effect of Gfi1 expression on NF-κB nuclear signaling, we found that LPS treatment enhances NF-κB DNA binding activity in Gfi1−/− macrophages as compared to Gfi1+/+ cells. Furthermore, over expression of Gfi1 protein resulted in negative regulation of NF-κB mediated gene activation in a dose-dependent manner. Chromatin immune precipitation with anti-p65 antibodies from LPS stimulated Gfi1+/+ and Gfi1−/− macrophages revealed enhanced NF-κB promoter occupancy at the TNF gene in Gfi1−/− macrophages as compared to Gfi1+/+ cells. In conclusion, our findings reveal a novel function for Gfi1 in the innate immune response by directly antagonizing NF-κB function. This molecular perceptive of TNF-α regulation during inflammation may provide an attractive strategy for therapeutic intervention in chronic inflammatory diseases and certain cancers.

B-cell development takes place in the bone marrow and is defined by a number of sequential steps that include the up-regulation of CD19, the rearrangement of immunoglobulin heavy and light chain genes (V(D)J recombination) and the expression of surface immunoglobulin. The early steps are regulated by cytokine signaling and the hierarchical expression of transcription factors, among them EBF, Pax5 and E2A and any interference with these critical elements leads to partial or total abrogation of B cell development. Here we present evidence that the POZ/BTB domain transcription factor Miz-1 (Zbtb17) represents an important novel regulator of the early development of follicular B cells. We have used gene targeting in mice to generate a non-functional allele of Miz-1 in all hematopoietic cells. In these mice, the development of adult follicular B cells is almost entirely abrogated, whereas the formation of marginal zone B-cells remain unaffected. Miz-1 deficiency correlated with the absence of CD19+ pro B-cells from the bone marrow and a block at the transition of the pre-pro-B cell to the pro-B cell stage. Although common lymphoid progenitors (CLPs) that are at the origin of B-cell development were present in Miz-1 deficient mice, they showed decreased expression of E2A, EBF and Pax5 compared to their wild type counterparts. Moreover, they were unable to differentiate in culture into more mature B cells even on stroma cells (OP9) and the presence or absence of IL-7. Interestingly, a forced expression of EBF or PAX5 in Miz-1 deficient progenitor cells did not rescue this phenotype. Furthermore, fetal B cell development, which has been shown to depend on EBF and Pax5, is not altered in Miz-1 deficient mice, suggesting that Miz-1 acts in a pathway that is independent of these critical B-cell regulators. In contrast, however, to EBF and Paxc5, the co-expression of a Bcl-2 transgene almost completely restored the development of more mature CD19+ or IgM+ B-cells in Miz-1 deficient mice. This indicated that Miz-1 is implicated in the regulation of cell survival at early stages of B cell development. Since it has been shown before that Bcl-2 is a downstream effector of Miz-1, it is conceivable that Miz-1 regulates Bcl-2 in the early B cell precursors, possibly as an element of the IL-7 signaling pathway, and thereby ensures their survival and proper development. We conclude that Miz-1 represents a novel regulator of early B cell development that exerts its function at a precise step in adult mice independently of other well-established regulators of B-cell development such as EBF or Pax5.