A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress.

Cells have the ability to sense, respond and adapt to environmental fluctuations. Stress causes a massive reorganization of the transcriptional program. Many examples of histone post-translational modifications (PTMs) have been associated with transcriptional activation or repression under steady-state growth conditions. Comparatively less is known about the role of histone PTMs in the cellular adaptive response to stress. Here, we performed high-throughput genetic screenings that provide a novel global map of the histone residues required for transcriptional reprogramming in response to heat and osmotic stress. Of note, we observed that the histone residues needed depend on the type of gene and/or stress, thereby suggesting a 'personalized', rather than general, subset of histone requirements for each chromatin context. In addition, we identified a number of new residues that unexpectedly serve to regulate transcription. As a proof of concept, we characterized the function of the histone residues H4-S47 and H4-T30 in response to osmotic and heat stress, respectively. Our results uncover novel roles for the kinases Cla4 and Ste20, yeast homologs of the mammalian PAK2 family, and the Ste11 MAPK as regulators of H4-S47 and H4-T30, respectively. This study provides new insights into the role of histone residues in transcriptional regulation under stress conditions.

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish.

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages.

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish.

BACKGROUND: Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated. DESIGN AND METHODS: TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro. RESULTS: Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis. CONCLUSIONS: This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).

Soluble inositol polyphosphates represent a variegate class of signalling molecules essential for the function of disparate cellular processes. Recently, the phytic acid derivate inositol pyrophosphate, InsP(7) (PP-IP(5) or IP(7)) has been shown to pyro-phosphorylate proteins in a kinase independent way. To begin to understand the functional importance of this new phosphorylation mechanism, a source of cold and radiolabelled InsP(7) is indispensable. However, cold InsP(7) is expensive to buy, and labelled InsP(7) is not commercially available. Here we provide a protocol to synthesise and purify InsP(7) to a level of purity required for in vivo and in vitro experiments. We begin by purifying recombinant mouse inositol hexakisphosphate kinase (IP6K1) from Escherichia coli. With purified IP6K1, we produce cold InsP(7) and 5beta[(32)P] InsP(7) that we subsequently use in vitro experiments to phosphorylate proteins extracts from different species.

Inositol pyrophosphates modulate hydrogen peroxide signalling.

Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.

Inositol pyrophosphates get the vip1 treatment.

Inositol pyrophosphates are unique signaling molecules implicated in the regulation of diverse cellular processes. Two new studies by Mulugu et al. (2007) and Lee et al. (2007) extend the biological and metabolic diversity of this class of molecules. They identify yeast Vip1 as a new inositol pyrophosphate synthase and show that the products of Vip1 activity regulate a cyclin/cyclin-dependent kinase complex.

RBMX gene is essential for brain development in zebrafish.

The human RBMX gene was discovered recently through its homology to the spermatogenesis candidate gene RBMY. Its position on the human X chromosome suggests that it may be involved in X-linked mental retardation syndromes. However, to date there is scant information on the in vivo role of RBMX. To address this issue, we have isolated a zebrafish rbmx orthologue and characterized its embryonic expression pattern. Zebrafish rbmx is maternally expressed and then widely expressed in the embryo up to 24 hr postfertilization. In later stages of embryonic development, rbmx transcripts are localized predominantly in the brain, branchial arches, and liver primordium. The function of rbmx during embryonic development was examined by the use of an antisense morpholino targeting rbmx. The rbmx-morphants displayed an underdeveloped head and eyes, reduced body size, defective somite patterning, and absence of jaws. Furthermore, in the absence of functional rbmx, expression of specific markers for the fore- and hindbrain (otx2, krox20) was severely reduced. These studies demonstrate for the first time that rbmx is required for normal embryonic development, in particular of the brain, consistent with a role in X-linked mental retardation.

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion.

OBJECTIVE: Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models. MATERIALS AND METHODS: To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors. RESULTS: Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages. CONCLUSION: These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

Harnessing zebrafish for the study of white blood cell development and its perturbation.

Considerable progress has been made in understanding the molecular basis of normal white blood cell development and its perturbation in disease through the use of clinical studies and traditional animal and cell line models. Despite this, however, many questions are still being answered and white blood cell disorders, including leukemia and lymphoma, remain a significant health problem. The zebrafish (Danio rerio) has emerged as a powerful alternative vertebrate model for the study of development and disease. We review the recent application of zebrafish to the study of white blood cell development and its disruption, particularly leukemogenesis. Such studies have highlighted the overall conservation of these processes throughout vertebrates, and establish zebrafish as a useful experimental model. This organism is now poised to make an important contribution to our understanding of the underlying genetic control of white blood cell development and its disruption, as well as the identification of new therapeutic agents.

The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish.

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.