Transcriptional regulation of the proto-oncogene Zfp521 by SPI1 (PU.1) and HOXC13.

The mouse zinc-finger gene Zfp521 (also known as ecotropic viral insertion site 3; Evi3; and ZNF521 in humans) has been identified as a B-cell proto-oncogene, causing leukemia in mice following retroviral insertions in its promoter region that drive Zfp521 over-expression. Furthermore, ZNF521 is expressed in human hematopoietic cells, and translocations between ZNF521 and PAX5 are associated with pediatric acute lymphoblastic leukemia. However, the regulatory factors that control Zfp521 expression directly have not been characterized. Here we demonstrate that the transcription factors SPI1 (PU.1) and HOXC13 synergistically regulate Zfp521 expression, and identify the regions of the Zfp521 promoter required for this transcriptional activity. We also show that SPI1 and HOXC13 activate Zfp521 in a dose-dependent manner. Our data support a role for this regulatory mechanism in vivo, as transgenic mice over-expressing Hoxc13 in the fetal liver show a strong correlation between Hoxc13 expression levels and Zfp521 expression. Overall these experiments provide insights into the regulation of Zfp521 expression in a nononcogenic context. The identification of transcription factors capable of activating Zfp521 provides a foundation for further investigation of the regulatory mechanisms involved in ZFP521-driven cell differentiation processes and diseases linked to Zfp521 mis-expression.

Histone h1 depletion impairs embryonic stem cell differentiation.

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

Cell-surface associated p43/endothelial-monocyte-activating-polypeptide-II in hepatocellular carcinoma cells induces apoptosis in T-lymphocytes.

OBJECTIVE: The novel, proinflammatory cytokine endothelial-monocyte-activating-polypeptide-II (EMAP-II) was first found in tumour cell supernatants and is closely related or identical to the p43 component of the mammalian multisynthetase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, including chemotactic, procoagulant and antiangiogenic properties. We recently showed that neoplastic but not normal hepatocytes expresses the 34-kDa molecule on the cell surface in vitro and the cell-surface expression is upregulated by treatment with tumour necrosis factor (TNF)-alpha/interferon (IFN)-gamma and/or hypoxia. We hypothesized an immune-regulatory role of EMAP-II within neoplastic tissues and investigated its effects on lymphocytes. METHODS: To study the role of EMAP-II in tumour cell-induced lymphocyte killing, Jurkat T-cells were co-cultured with a range of hepatocellular carcinoma (HCC) cell monolayers (HuH-7, HepG2 and Alexander cells), which were either untreated or treated with TNF-alpha/IFN-gamma under normoxic and hypoxic conditions over a period of 16-24 hours. Flow cytometric analysis of apoptosis in Jurkat cells was performed using the annexin-V-FITC/propidium iodide technique. RESULTS: rEMAP-II caused a dose-dependent apoptosis in Jurkat T-cells. Co-culture of Jurkat cells with HCC cell monolayers induced significant apoptosis of the Jurkat cells. In general, under normoxic conditions, cytokine-treated HCC cell monolayer caused more apoptosis than untreated cells. This effect was enhanced by hypoxia. Critically, native EMAP-II expressed on the surface of the HCC cells also induced activation of caspase-8 and apoptosis in Jurkat cells, which was partially but significantly blocked by addition of polyclonal antibodies against EMAP-II to the incubation mixture. CONCLUSION: Our data suggest that membrane-bound EMAP-II is cytotoxic to lymphocytes and, therefore, might constitute a component of a novel, immunosuppressive pathway by which HCC cells may eliminate attacking T-cells and evade the immune system. The mechanism by which it does so is currently under investigation.