SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development.

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.

Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability.

Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. The possibility of manipulating the protein content by using protein depletion procedures makes egg extract a powerful system to study proteins whose inactivation results in cellular lethality. The egg extract has been increasingly used to study DNA damage response and the coordination of DNA replication with DNA repair. The recent development of advanced imaging techniques based on electron microscopy has allowed the characterization of replication intermediates formed in the absence of essential DNA repair proteins. These studies have been important to understand how cells maintain genome stability under unchallenged and stressful conditions. Here, we present a collection of protocols that have been developed to recapitulate DNA damage response activated by chromosome breakage in egg extract and to isolate replication intermediates for electron microscopy analysis using sperm nuclei or more defined genomic substrates.

Nucleophosmin leukemogenic mutant activates Wnt signaling during zebrafish development.

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein with both oncogenic and tumor suppressor functions. Mutations of the NPM1 gene are the most frequent genetic alterations in acute myeloid leukemia (AML) and result in the expression of a mutant protein with aberrant cytoplasmic localization, NPMc+. Although NPMc+ causes myeloproliferation and AML in animal models, its mechanism of action remains largely unknown. Here we report that NPMc+ activates canonical Wnt signaling during the early phases of zebrafish development and determines a Wnt-dependent increase in the number of progenitor cells during primitive hematopoiesis. Coherently, the canonical Wnt pathway is active in AML blasts bearing NPMc+ and depletion of the mutant protein in the patient derived OCI-AML3 cell line leads to a decrease in the levels of active ß-catenin and of Wnt target genes. Our results reveal a novel function of NPMc+ and provide insight into the molecular pathogenesis of AML bearing NPM1 mutations.

Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.

Monoclonal antibodies isolated by large-scale screening are suitable for labeling adult zebrafish (Danio rerio) tissues and cell structures.

The lack of a sufficient number of antibodies represents an obstacle in the research performed using the zebrafish (Danio rerio) as a model organism. On the other hand, high-throughput generation of antibodies, especially those suitable for immunohistochemistry, is not an established methodology. Here we present the results of an immunization experiment with a zebrafish tissue lysate that allowed for the isolation of hundreds of monoclonal antibodies suitable for labeling of a large variety of zebrafish tissue and cell structures. Some of them were further characterized in terms of detailed localization and age-dependent expression. In addition, the antigen recognized by one of them was first immunoprecipitated and then identified by mass spectrometry. Furthermore, immunofluorescence-competent recombinant antibodies were also isolated by panning large repertoire phage display libraries, in both single-chain (scFv) and single-domain (VHH) format. Such selection alternative is simpler to organize and could contribute to limit the costs of antibody screening and production.

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells.

Constitutively active, 'oncogenic' H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.

The tumor suppressor PRDM5 regulates Wnt signaling at early stages of zebrafish development.

PRDM genes are a family of transcriptional regulators that modulate cellular processes such as differentiation, cell growth and apoptosis. Some family members are involved in tissue or organ maturation, and are differentially expressed in specific phases of embryonic development. PRDM5 is a recently identified family member that functions as a transcriptional repressor and behaves as a putative tumor suppressor in different types of cancer. Using gene expression profiling, we found that transcriptional targets of PRDM5 in human U2OS cells include critical genes involved in developmental processes, and specifically in regulating wnt signaling. We therefore assessed PRDM5 function in vivo by performing loss-of-function and gain-of-function experiments in zebrafish embryos. Depletion of prdm5 resulted in impairment of morphogenetic movements during gastrulation and increased the occurrence of the masterblind phenotype in axin+/- embryos, characterized by the loss of eyes and telencephalon. Overexpression of PRDM5 mRNA had opposite effects on the development of anterior neural structures, and resulted in embryos with a shorter body axis due to posterior truncation, a bigger head and abnormal somites. In situ hybridization experiments aimed at analyzing the integrity of wnt pathways during gastrulation at the level of the prechordal plate revealed inhibition of non canonical PCP wnt signaling in embryos overexpressing PRDM5, and over-activation of wnt/beta-catenin signaling in embryos lacking Prdm5. Our data demonstrate that PRDM5 regulates the expression of components of both canonical and non canonical wnt pathways and negatively modulates wnt signaling in vivo.