The Contribution of the Zebrafish Model to the Understanding of Polycomb Repression in Vertebrates.

Polycomb group (PcG) proteins are highly conserved proteins assembled into two major types of complexes, PRC1 and PRC2, involved in the epigenetic silencing of a wide range of gene expression programs regulating cell fate and tissue development. The crucial role of PRC1 and PRC2 in the fundamental cellular processes and their involvement in human pathologies such as cancer attracted intense attention over the last few decades. Here, we review recent advancements regarding PRC1 and PRC2 function using the zebrafish model. We point out that the unique characteristics of the zebrafish model provide an exceptional opportunity to increase our knowledge of the role of the PRC1 and PRC2 complexes in tissue development, in the maintenance of organ integrity and in pathology.

PRC1 components exhibit different binding kinetics in Polycomb bodies.

BACKGROUND INFORMATION: Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined. RESULTS: Fluorescently tagged PcG proteins CBXs and their partners BMI1 and RING1 form foci of different sizes and intensities in human U2OS cells. Fluorescence recovery after photobleaching (FRAP) analysis reveals that PcG dynamics outside foci is governed by diffusion as complexes and transient binding. In sharp contrast, recovery curves inside foci are substantially slower and exhibit large variability. The slow binding component amplitudes correlate with the intensities and sizes of these foci, suggesting that foci contained varying numbers of binding sites. CBX4-green fluorescent protein (GFP) foci were more stable than CBX8-GFP foci; yet the presence of CBX4 or CBX8-GFP in the same focus had a minor impact on BMI1 and RING1 recovery kinetics. CONCLUSION: We propose that FRAP recovery curves provide information about PcG binding to their target genes outside foci and about PcG spreading onto chromatin inside foci.

Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells.

Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING1/RNF2, PHC, and BMI1/PCGF families. Mammalian genomes encode five CBX family members (CBX2, CBX4, CBX6, CBX7, and CBX8) that are believed to have distinct biological functions. Here, we applied a tandem affinity purification (TAP) approach coupled with tandem mass spectrometry (MS/MS) methodologies in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX family proteins are mutually exclusive and define distinct PRC1-like protein complexes. CBX proteins also interact with different efficiencies with the other PRC1 components. Among the novel CBX interacting partners, protein kinase 2 associates with all CBX-PRC1 protein complexes, whereas 14-3-3 proteins specifically bind to CBX4. 14-3-3 protein binding to CBX4 appears to modulate the interaction between CBX4 and the BMI1/PCGF components of PRC1, but has no effect on CBX4-RING1/RNF2 interaction. Finally, we suggest that differences in CBX protein interactions would account, at least in part, for distinct subnuclear localization of the CBX family members.

Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells.

BACKGROUND: CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. METHODS: After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. RESULTS: We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. CONCLUSION: Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

Analysis of the human HP1 interactome reveals novel binding partners.

Heterochromatin protein 1 (HP1) has first been described in Drosophila as an essential component of constitutive heterochromatin required for stable epigenetic gene silencing. Less is known about the three mammalian HP1 isotypes CBX1, CBX3 and CBX5. Here, we applied a tandem affinity purification approach coupled with tandem mass spectrometry methodologies in order to identify interacting partners of the mammalian HP1 isotypes. Our analysis identified with high confidence about 30-40 proteins co-eluted with CBX1 and CBX3, and around 10 with CBX5 including a number of novel HP1-binding partners. Our data also suggest that HP1 family members are mainly associated with a single partner or within small protein complexes composed of limited numbers of components. Finally, we showed that slight binding preferences might exist between HP1 family members.

Loss of Polycomb Repressive Complex 2 Function Alters Digestive Organ Homeostasis and Neuronal Differentiation in Zebrafish.

Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of eed, a gene coding for an essential component of the PRC2. Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10-12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.

Vimentin Promotes the Aggressiveness of Triple Negative Breast Cancer Cells Surviving Chemotherapeutic Treatment.

Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.

Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP-calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP-MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.

The Polycomb Orthologues in Teleost Fishes and Their Expression in the Zebrafish Model.

The Polycomb Repressive Complex 1 (PRC1) is a chromatin-associated protein complex involved in transcriptional repression of hundreds of genes controlling development and differentiation processes, but also involved in cancer and stem cell biology. Within the canonical PRC1, members of Pc/CBX protein family are responsible for the targeting of the complex to specific gene loci. In mammals, the Pc/CBX protein family is composed of five members generating, through mutual exclusion, different PRC1 complexes with potentially distinct cellular functions. Here, we performed a global analysis of the cbx gene family in 68 teleost species and traced the distribution of the cbx genes through teleost evolution in six fish super-orders. We showed that after the teleost-specific whole genome duplication, cbx4, cbx7 and cbx8 are retained as pairs of ohnologues. In contrast, cbx2 and cbx6 are present as pairs of ohnologues in the genome of several teleost clades but as singletons in others. Furthermore, since zebrafish is a widely used vertebrate model for studying development, we report on the expression of the cbx family members during zebrafish development and in adult tissues. We showed that all cbx genes are ubiquitously expressed with some variations during early development.

[Cancer cell transplantation in zebrafish: From translational research to personalized medicine]. / La transplantation de cellules tumorales chez le poisson zèbre : de la recherche translationnelle à la médecine personnalisée.

Primarily used in genetic studies of development, the zebrafish (Danio rerio) has rapidly emerged as a promising animal model of human cancer. Cancer cell transplantation in zebrafish constitutes a key platform for clinical research since it allows to study cellular and molecular events involved in various aspects of tumorigenesis and to evaluate the efficacy of therapeutic molecules in vivo. Applied to patient-derived cells, the xenotransplantation approach in zebrafish allows to define the most appropriate therapeutic strategies for specific alterations found in patients in the context of personalized medicine. This review discusses the zebrafish transplantation model for the study of cancer development and drug discovery.

Genetic Engineering of Zebrafish in Cancer Research.

Zebrafish (Danio rerio) is an excellent model to study a wide diversity of human cancers. In this review, we provide an overview of the genetic and reverse genetic toolbox allowing the generation of zebrafish lines that develop tumors. The large spectrum of genetic tools enables the engineering of zebrafish lines harboring precise genetic alterations found in human patients, the generation of zebrafish carrying somatic or germline inheritable mutations or zebrafish showing conditional expression of the oncogenic mutations. Comparative transcriptomics demonstrate that many of the zebrafish tumors share molecular signatures similar to those found in human cancers. Thus, zebrafish cancer models provide a unique in vivo platform to investigate cancer initiation and progression at the molecular and cellular levels, to identify novel genes involved in tumorigenesis as well as to contemplate new therapeutic strategies.

Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions.

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours.

BACKGROUND: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks. RESULTS: To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin. CONCLUSION: FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.

Ezh1 arises from Ezh2 gene duplication but its function is not required for zebrafish development.

Trimethylation on H3K27 mediated by Polycomb Repressive Complex 2 (PRC2) is required to control gene repression programs involved in development, regulation of tissue homeostasis or maintenance and lineage specification of stem cells. In Drosophila, the PRC2 catalytic subunit is the single protein E(z), while in mammals this function is fulfilled by two proteins, Ezh1 and Ezh2. Based on database searches, we propose that Ezh1 arose from an Ezh2 gene duplication that has occurred in the common ancestor to elasmobranchs and bony vertebrates. Expression studies in zebrafish using in situ hybridization and RT-PCR followed by the sequencing of the amplicon revealed that ezh1 mRNAs are maternally deposited. Then, ezh1 transcripts are ubiquitously distributed in the entire embryo at 24 hpf and become more restricted to anterior part of the embryo at later developmental stages. To unveil the function of ezh1 in zebrafish, a mutant line was generated using the TALEN technology. Ezh1-deficient mutant fish are viable and fertile, but the loss of ezh1 function is responsible for the earlier death of ezh2 mutant larvae indicating that ezh1 contributes to zebrafish development in absence of zygotic ezh2 gene function. Furthermore, we show that presence of ezh1 transcripts from the maternal origin accounts for the delayed lethality of ezh2-deficient larvae.

[The zebrafish model in oncology]. / Le modèle poisson zèbre dans la lutte contre le cancer.

Although cell culture and mouse models will remain a cornerstone of cancer research, the unique capabilities of the zebrafish outline the potential of this model for shedding light on cancer biology in vivo. Zebrafish develops cancers spontaneously, after chemical mutagenesis or through genetic manipulations. Furthermore, zebrafish cancers are similar to human tumors at the histological and molecular levels allowing the study of tumor initiation, progression and heterogeneity. Xenotransplantation of human cancer cells in embryos or adult zebrafish presents the advantage of following cancer cell behavior in vivo. Finally, zebrafish embryos are used in molecule screens and contribute to the identification of novel anti-cancer therapeutic strategies. Here, we review different involvements of the zebrafish model in cancer research.

Combining genotypic and phenotypic analyses on single mutant zebrafish larvae.

Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [[2], [3]]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae. •The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration.•The technique is optimized in order to be applied on single zebrafish embryos and larvae.

The control of histone lysine methylation in epigenetic regulation.

Histone lysine methylation plays a fundamental role in chromatin organization and function. This epigenetic mark is involved in many biological processes such as heterochromatin formation, chromosome X inactivation, genomic imprinting and transcriptional regulation. Here, we review recent advances in how histone lysine methylation participates in these biological events, and the enzymes that control histone lysine methylation and demethylation.

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish.

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2-deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2-deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2-deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.

The Polycomb Group Protein Pcgf1 Is Dispensable in Zebrafish but Involved in Early Growth and Aging.

Polycomb Repressive Complex (PRC) 1 regulates the control of gene expression programs via chromatin structure reorganization. Through mutual exclusion, different PCGF members generate a variety of PRC1 complexes with potentially distinct cellular functions. In this context, the molecular function of each of the PCGF family members remains elusive. The study of PCGF family member expression in zebrafish development and during caudal fin regeneration reveals that the zebrafish pcgf genes are subjected to different regulations and that all PRC1 complexes in terms of Pcgf subunit composition are not always present in the same tissues. To unveil the function of Pcgf1 in zebrafish, a mutant line was generated using the TALEN technology. Mutant pcgf1-/- fish are viable and fertile, but the growth rate at early developmental stages is reduced in absence of pcgf1 gene function and a significant number of pcgf1-/- fish show signs of premature aging. This first vertebrate model lacking Pcgf1 function shows that this Polycomb Group protein is involved in cell proliferation during early embryogenesis and establishes a link between epigenetics and aging.

Diverse involvement of EZH2 in cancer epigenetics.

EZH2 is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2) which catalyzes methylation of histone H3 at lysine 27 (H3K27me) and mediates gene silencing of target genes via local chromatin reorganization. Numerous evidences show that EZH2 plays a critical role in cancer initiation, progression and metastasis, as well as in cancer stem cell biology. Indeed, EZH2 dysregulation alters gene expression programs in various cancer types. The molecular mechanisms responsible for EZH2 alteration appear to be diverse and depending on the type of cancer. Furthermore, accumulating evidences indicate that EZH2 could also act as a PRC2-independent transcriptional activator in cancer. In this review, we address the current understanding of the oncogenic role of EZH2, including the mechanisms of EZH2 dysregulation in cancer and progresses in therapeutic approaches targeting EZH2.