cis-Regulation and chromatin dynamics of the hbox12 gene during the embryogenesis of Paracentrotus lividus.

The GRN specifying the dorsal-ventral (D-V) axis of the sea urchin embryo is currently under investigation. An early input for D-V polarity is given by a redox gradient probably generated by an asymmetrical distribution of maternal mitochondria (1). Only on the future ventral side, the oxidizing environment induces the expression of the nodal gene, an essential regulator of D-V polarization (2). By contrast, on the future dorsal side, a reducing environment activates the hypoxia inducible factor (HIF-1α) (3). The hbox12 transcription repressor is an early marker of the dorsal side of the embryo, in which it negatively regulates the expression of nodal (4, 5). Interestingly, by in silico ana…

Functional studies of regulatory genes in the sea urchin embryo.

Histones, Their Variants and Post-translational Modifications in Zebrafish Development.

Complex multi-cellular organisms are shaped starting from a single-celled zygote, owing to elaborate developmental programs. These programs involve several layers of regulation to orchestrate the establishment of progressively diverging cell type-specific gene expression patterns. In this scenario, epigenetic modifications of chromatin are central in influencing spatiotemporal patterns of gene transcription. In fact, it is generally recognized that epigenetic changes of chromatin states impact on the accessibility of genomic DNA to regulatory proteins. Several lines of evidence highlighted that zebrafish is an excellent vertebrate model for research purposes in the field of developmental ep…

Dorsal/Ventral asymmetric expression of nodal in the early sea urchin embryo relies on specific suppression in dorsal cells by the Hbox12 homeodomain-containing regulator

Dorsal/Ventral (DV) polarization of the sea urchin embryo is directed by a Nodal-expressing signaling centre located on the ventral side. The initial breaking of the symmetry and positioning of the organizer are unclear. We show that, in Paracentrotus lividus embryos, the Hbox12 homeodomaincontaining repressor is expressed on the opposite side and precedes the onset of nodal transcription. Hbox12 misexpression provokes DV abnormalities, attenuating nodal and nodal-dependent gene transcription. Reciprocally, clonal hbox12 loss-of-function imposed by blastomere transplantation or gene transfer assays disrupts DV polarization and allows ectopic expression of nodal. Remarkably, the localized kn…

Dinamica della cromatina nella regolazione del gene con homeobox Hbox12 in P. lividus.

Identificazione dei geni codificanti per i recettori di acido y-amminobutirrico nel genoma del riccio di mare

Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo

Dorsal/ventral (DV) patterning of the sea urchin embryo relies on a ventrally-localized organizer expressing Nodal, a pivotal regulator of the DV gene regulatory network. However, the inceptive mechanisms imposing the symmetry-breaking are incompletely understood. In Paracentrotus lividus, the Hbox12 homeodomain-containing repressor is expressed by prospective dorsal cells, spatially facing and preceding the onset of nodal transcription. We report that Hbox12 misexpression provokes DV abnormalities, attenuating nodal and nodal-dependent transcription. Reciprocally, impairing hbox12 function disrupts DV polarity by allowing ectopic expression of nodal. Clonal loss-of-function, inflicted by b…

Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo

Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 …

Ruolo delle HDAC sul silenziamento genico nell’embrione di riccio di mare

Hbox12: an old gene with a new function.

SEA URCHIN RESEARCH: MILESTONES, MEMORIES, AND FUTURE CHALLENGES

The sea urchin eggs and embryos have been used for nearly two centuries as experimental models for classical and modern developmental biology. In the late 1870s, the ground-breaking observations independently obtained by Hertwig and Fol highlighted for the first time that a single sperm enters the oocyte and the male and female pronuclei fuse at fertilization.1 From that point on, the seminal studies of Boveri, Driesch, and Herbst allowed conceptualization of basic biological themes, such as the chromosome theory of heredity.2 In the first half of the twentieth century, the embryo manipulation experiments performed by Hörstadius and Runnström further advanced the field, introducing the conc…

The Ambulacraria-specific COMPASS-like protein contributes to sns5 chromatin insulator function in the sea urchin embryo

Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin α-H2A Histone Gene

The tandemly repeated sea urchin alpha-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the alpha-H2A gene depends on the binding of the MBF-1 activator to the 5' enhancer, while down-regulation relies on the functional interaction between the 3' sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Her…

Evaluation of Epigenetic and Radiomodifying Effects during Radiotherapy Treatments in Zebrafish

Radiotherapy is still a long way from personalizing cancer treatment plans, and its effectiveness depends on the radiosensitivity of tumor cells. Indeed, therapies that are efficient and successful for some patients may be relatively ineffective for others. Based on this, radiobiological research is focusing on the ability of some reagents to make cancer cells more responsive to ionizing radiation, as well as to protect the surrounding healthy tissues from possible side effects. In this scenario, zebrafish emerged as an effective model system to test for radiation modifiers that can potentially be used for radiotherapeutic purposes in humans. The adoption of this experimental organism is fu…

Long non-coding RNAs during embryogenesis of the sea urchin

Intriguing studies suggest that long non-coding RNAs (lncRNAs) tend to act in cis with neighboring protein-coding genes. Based on this notion, we assessed whether lncRNAs originate from adjacent genomic regions of selected protein-coding genes, such as early-H2A histone and strim1, that play important functions during development of the sea urchin Paracentrotus lividus. Downregulation of H2A expression relies on the sns5 chromatin insulator placed at the 3'-end of H2A. Northern blot hybridization show that a lncRNA (of ~600 nt), not overlapping with the H2A mRNA, is transcribed from sns5, following H2A expression. The strim1 locus encodes the first echinoderm TRIM-containing factor which fu…

Environmental epigenetics in zebrafish

Abstract It is widely accepted that the epigenome can act as the link between environmental cues, both external and internal, to the organism and phenotype by converting the environmental stimuli to phenotypic responses through changes in gene transcription outcomes. Environmental stress endured by individual organisms can also enforce epigenetic variations in offspring that had never experienced it directly, which is termed transgenerational inheritance. To date, research in the environmental epigenetics discipline has used a wide range of both model and non-model organisms to elucidate the various epigenetic mechanisms underlying the adaptive response to environmental stimuli. In this rev…

Model organisms and their application in environmental epigenetics

Abstract Epigenetic mechanisms operate at the interface between the environment and the genome of a given organism. More particularly, they convert environmental stimuli as diverse as chemical pollutants, nutritional components, temperature changes, and social conditions into changes in the chromatin landscape, which influence gene expression in the absence of alterations in the DNA sequence, ultimately leading to phenotype adaptation and diversification. The use of experimental model organisms in epigenetics research allows for the careful manipulation of environmental factors in order to understand how they mechanistically modulate the establishment, maintenance, and inheritance of epigen…

cis-Regulatory Analysis of the homeobox- containing gene hbox12

Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5' and 3' regions and including the enhancer blocker sns.

The tandem repeated sea urchin alpha-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing …

Novel Translational Read-through–Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS…

Symmetry Breaking and Establishment of Dorsal/Ventral Polarity in the Early Sea Urchin Embryo

The mechanisms imposing the Dorsal/Ventral (DV) polarity of the early sea urchin embryo consist of a combination of inherited maternal information and inductive interactions among blastomeres. Old and recent studies suggest that a key molecular landmark of DV polarization is the expression of nodal on the future ventral side, in apparent contrast with other metazoan embryos, where nodal is expressed dorsally. A subtle maternally-inherited redox anisotropy, plus some maternal factors such as SoxB1, Univin, and p38-MAPK have been identified as inputs driving the spatially asymmetric transcription of nodal. However, all the mentioned factors are broadly distributed in the embryo as early as no…

The multiplicity of Argonaute complexes in mammalian cells

Argonautes (AGOs) are a highly conserved family of proteins found in most Eukaryotes, and involved in mechanisms of gene regulation, both at the transcriptional and post-transcriptional level. Among other functions, AGO proteins associate with microRNAs to mediate the post-transcriptional repression of protein-coding genes. In this process, AGOs associate with members of the trinucleotide repeat containing 6 protein (TNRC6) protein family to form the core of the RNA-induced silencing complex (RISC), the effector machinery that mediates microRNA function. However, the description of the exact composition of the RISC has been a challenging task due to the fact the AGO's interactome is dynamic…

LA DINAMICA DELLA CROMATINA NELLA REGOLAZIONE DELL’ESPRESSIONEGENICA DELL’EMBRIONE DI RICCIO DI MARE

Specificazione dell'asse Dorso/Ventrale dell'embrione di riccio di mare.

The sea urchin embryo: a model to study Alzheimer's beta amyloid induced toxicity.

Abstract Alzheimer’s disease (AD) is the most common form of dementia. The cause of AD is closely related to the accumulation of amyloid beta peptide in the neuritic plaques. The use of animal model systems represents a good strategy to elucidate the molecular mechanism behind the development of this pathology. Here we use the Paracentrotus lividus embryo to identify molecules and pathways that can be involved in the degenerative process. As a first step, we identified the presence of an antigen related to the human APP, called Pl APP. This antigen, after gastrula stage, is processed producing a polypeptide of about 10 kDa. By immunohistochemistry we localized the Pl APP antigen in some ser…

Computational prediction of the Helitron-N2 (HeN2) transposable element in the strim1 locus of the Paracentrotus lividus genome

Transposons constitute a significant component of the eukaryotic genome. Among others, Helitrons represent a novel major class of eukaryotic transposons, and are fundamentally different from classical ones in terms of their structure and mechanism of transposition [1]. In particular, Helitrons constitute ~1% of the sea urchin genome [2]. By a in silico approach focused on the genome of the Mediterranean sea urchin Paracentrotus lividus, we have predicted regions of high sequence identity to a Helitron-N2 (HeN2) element in the strim1 locus. Of interest, HeN2 sequences lies within the 5’ non coding region of the strim1a and strim1b genes, spanning from -105 to -2255 (the start codon ATG of st…

Spatially restricted expression of strim, a novel TRIM-containing gene, in the oral ectoderm of the developing sea urchin embryo Paracentrotus lividus.

The recent wealth of genomic data has confirmed that bilaterians as simple as nematodes and as complex as humans use the same basic toolkit of transcription factors and signaling molecules to process spatial and temporal information during development (1,2). Among these regulators, the TRIM/RBCC (Tripartite motif/RING-Bbox-Coiled Coil) family represents one of the largest classes of putative E3 ubiquitin ligases that play essential roles in cell cycle regulation and development (3). It consists of rapidly evolving genes, identified in eukaryotic organisms (4,5). In the sea urchin Paracentrotus lividus we have identified a locus containing two copies, probably originated from a duplication e…

Suppression of nodal expression in prospective dorsal cells of the early sea urchin embryo by the hbox12 homeodomain regulator

Dorsal/Ventral (DV) axis formation in the sea urchin embryo depends upon the expression of nodal on the ventral side, which behaves as a DV organizing centre. However, only fuzzy clues are known as to the early symmetry-breaking steps that lead to the positioning of such an organizer. An extremely interesting candidate for this role is the hbox12 homeobox-containing gene. In Paracentrotus lividus, hbox12 expression is antecedent and complementary with respect to that of nodal, being confined in prospective dorsal cells. We show that ectopic expression of Hbox12 provokes DV abnormalities and attenuates nodal as well as nodal-dependent gene transcription. By blastomere transplantation, we als…

Localized expression of Strim1, a novel member of the TRIM-containing family, guides the skeletal morphogenetic program of the sea urchin embryo

The building of the skeleton in the indirect developing sea urchin embryo is a complex morphogenetic process that is executed by the Primary Mesenchyme Cells or PMCs (Ettensohn et al, 1997; Wilt 2002). It is well known that the PMCs acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth (Armstrong 1993; Cavalieri et al, 2003; Röttinger et al, 2008). In this study, we analyze the function of a novel gene, encoding for a tripartite motif-containing (TRIM) protein denoted strim1, that adds up to the list of genes constituting the epithelial-mesenchymal signaling network. We show that strim1 is expressed in ectoderm regions adjacen…

Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 a-histone genes is modulated by enhancer and chromatin insulator

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) alpha-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence su…

Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos

AbstractIn the sea urchin embryo skeletogenesis is the result of a complex series of molecular and cellular events that coordinate the morphogenetic process. Past and recent evidence strongly indicate that skeletal initiation and growth are strictly dependent on signals emanating from the oral ectodermal wall. As previously suggested, Orthopedia (Otp), a homeodomain-containing transcription factor specifically expressed in a small subset of oral ectoderm cells, might be implicated in this signalling pathway. In this study, we utilize three different strategies to address the issue of whether Otp is an upstream regulator of sketelogenesis. We describe the effects of microinjection of Otp mor…

Diversification of spatiotemporal expression and copy number variation of the echinoid hbox12/pmar1/micro1 multigene family

Changes occurring during evolution in the cis-regulatory landscapes of individual members of multigene families might impart diversification in their spatiotemporal expression and function. The archetypal member of the echinoid hbox12/pmar1/micro1 family is hbox12-a, a homeobox-containing gene expressed exclusively by dorsal blastomeres, where it governs the dorsal/ventral gene regulatory network during embryogenesis of the sea urchin Paracentrotus lividus. Here we describe the inventory of the hbox12/pmar1/micro1 genes in P. lividus, highlighting that gene copy number variation occurs across individual sea urchins of the same species. We show that the various hbox12/pmar1/micro1 genes grou…

Perturbation of Developmental Regulatory Gene Expression by a G-Quadruplex DNA Inducer in the Sea Urchin Embryo.

The G-quadruplex (G4) is a four-stranded DNA structure identified in vivo in guanine-rich regions located in the promoter of a number of genes. Intriguing evidence suggested that small molecules acting as G4-targeting ligands could potentially regulate multiple cellular processes via either stabilizing or disruptive effects on G4 motifs. Research in this field aims to prove the direct role of G4 ligands and/or structures on a specific biological process in a complex living organism. In this study, we evaluate in vivo the effects of a nickel(II)-salnaphen-like complex, named Nisaln, a potent G4 binder and stabilizer, during embryogenesis of the sea urchin embryo. We describe developmental de…

Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-…

GENE REGULATORY NETWORK OF THE EARLY SEA URCHIN EMBRYO INVOLVES THE HBOX12 HOMEODOMAIN-CONTAINING REGULATOR

Editorial: Zebrafish Epigenetics.

A key area of focus in the field of epigenetics pertains the comprehension of the functional relevance of the epigenetic mechanisms occurring during embryogenesis to shape normal developmental trajectories and adult phenotypes (Atlasi and Stunnenberg, 2017; Skvortsova et al., 2018; Cavalieri, 2021; Marchione et al., 2021). Several lines of evidence highlighted that the small freshwater cyprinid Danio rerio, commonly known as zebrafish, is an excellent vertebrate model for research purposes in the field of epigenetics (Huang et al., 2013; Balasubramanian et al., 2019; Horsfield, 2019; Cavalieri, 2020). The general strengths of zebrafish over concurrent models are well known: ease of husbandr…

Author response: Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo

Il gene con homeobox P1Hbox12 è coinvolto nella specificazione dell’endo-mesoderma dell’embrione di riccio di mare Paracentrotus lividus

EFFECT OF Γ-AMINOBUTYRRIC ACID (GABA) EXPOSURE ON EMBRYOGENESIS OF PARACENTROTUS LIVIDUS AND IDENTIFICATION OF GABA-RECEPTOR GENES IN SEA URCHINS

Developmental processes are controlled by regulatory genes encoding for transcription factors and signaling molecules. Functional relationships between these genes are described by gene regulatory networks (GRN), models which allow integration of various levels of information. The sea urchin embryo is an experimental model system which offers many advantages for the analysis of GRN. Recently, the GRN that governs the biomineralization of the sea urchin embryonic skeleton has begun to be deciphered. Preliminary evidence suggest that the γ- aminobutyric acid (GABA) signaling pathway is involved in skeletal morphogenesis during development of the sea urchin. GABA is a molecule synthesized by n…

Epigenetic Modulation of Chromatin States and Gene Expression by G-Quadruplex Structures

G-quadruplexes are four-stranded helical nucleic acid structures formed by guanine-rich sequences. A considerable number of studies have revealed that these noncanonical structural motifs are widespread throughout the genome and transcriptome of numerous organisms, including humans. In particular, G-quadruplexes occupy strategic locations in genomic DNA and both coding and noncoding RNA molecules, being involved in many essential cellular and organismal functions. In this review, we first outline the fundamental structural features of G-quadruplexes and then focus on the concept that these DNA and RNA structures convey a distinctive layer of epigenetic information that is critical for the c…

Identification of a putative regulator of early-histone gene expression of Paracentrotus lividus

Drosophila GAGA factor (GAF) is a nuclear protein, conserved along evolution, with multiple roles in gene regulation, chromatin remodelling, Polycomb-dependent silencing, and insulator functions (1). GAF recognizes and specifically binds GAGAG consensus DNA motif by its C2H2-type zinc-finger domain, and interacts with other regulatory factors by its BTB/POZ domain. We have identified plGaga, a cDNA coding for a putative GAF of the sea urchin P. lividus, which showed significant sequence similarity with Drosophila and vertebrate GAFs. Real time RT-PCR revealed that the plGaga RNA is always present during embryo development decreasing rapidly in abundance at the larval pluteus stage. We raise…

The echinoid hbox12 multigene family: copy number variation and spatiotemporal expression

In silico identification of the TRIM gene complement of the sea urchin and computational prediction of Helitron-N2 transposable element in the 5’ non-coding region of two strim1 genes

Non-primate lentiviral vectors and their applications in gene therapy for ocular disorders

Lentiviruses have a number of molecular features in common, starting with the ability to integrate their genetic material into the genome of non-dividing infected cells. A peculiar property of non-primate lentiviruses consists in their incapability to infect and induce diseases in humans, thus providing the main rationale for deriving biologically safe lentiviral vectors for gene therapy applications. In this review, we first give an overview of non-primate lentiviruses, highlighting their common and distinctive molecular characteristics together with key concepts in the molecular biology of lentiviruses. We next examine the bioengineering strategies leading to the conversion of lentiviruse…

Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells.

Abstract PlOtp (Orthopedia), a homeodomain-containing transcription factor, has been recently characterized as a key regulator of the morphogenesis of the skeletal system in the embryo of the sea urchin Paracentrotus lividus . Otp acts as a positive regulator in a subset of oral ectodermal cells which transmit short-range signals to the underlying primary mesenchyme cells where skeletal synthesis is initiated. To shed some light on the molecular mechanisms involved in such a process, we begun a functional analysis of the cis -regulatory sequences of the Otp gene. Congruent with the spatial expression profile of the endogenous Otp gene, we found that while a DNA region from −494 to +358 is s…

Ectoderm restricted expression of the TRIM-containing factor Strim1 directs the migration, aggregation and terminal differentiation of mesenchymal cells during skeletal morphogenesis of the sea urchin embryo

Genome wide mapping of the MBF-1 binding sites during embryogenesis of the sea urchin reveals it is a chromatin organizer.

The Zinc-finger MBF1 factor is a transcription activator involved in the expression of the early histone genes during development of the sea urchin embryo (1). The DNA-binding domain of MBF1 shares high sequence similarity with that of the CTCF chromatin organizer but, unexpectedly, extensive in silico analysis failed to identify the sea urchin CTCF ortholog (2, 3). This led us to speculate that MBF1 could have co-opted the function of CTCF during evolution of the echinoderms. To support this hypothesis, we performed the genome-wide MBF1-binding sites mapping in the P. lividus genome, by chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq). We observed that MBF1 bi…

Inducible and reversible inhibition of mirna-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by invest…

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by invest…

The sea urchin histone H2A enhancer-binding protein MBF-1 is needed for maximal expression also for the H3 gene, while is buffered by the sns5 insulator on its interaction with the downstream H1 promoter.

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator bin…

The sea urchin sns5 chromatin insulator settles a gene therapy vector into an independent domain of expression in the vertebrate genome

One of the critical aspects of introducing a transgene into the eukaryotic genome is the great variability of gene expression due to position effects (1). Chromatin-dependent repressive states could be overcome by incorporation in the transgene of chromatin insulators, functioning to establish and delimit domains of expression. We have previously demonstrated that the sea urchin sns5 DNA element has the typical features of an insulator: by acting as enhancer blocker, it shields promoters from neighboring regulatory elements, and by acting as barrier it buffers a transgene from the propagation of condensed chromatin (2,3). We have investigated the use of sns5 in the field of gene therapy. Ou…

High-Intensity Focused Ultrasound- and Radiation Therapy-Induced Immuno-modulation: Comparison and Potential Opportunities

In recent years, high-intensity focused ultrasound (HIFU) has emerged as a new and promising non-invasive and non-ionizing ablative technique for the treatment of localized solid tumors. Extensive pre-clinical and clinical studies have evidenced that, in addition to direct destruction of the primary tumor, HIFU-thermoablation may elicit long-term systemic host anti-tumor immunity. In particular, an important consequence of HIFU treatment includes the release of tumor-associated antigens (TAAs), the secretion of immuno-suppressing factors by cancer cells and the induction of cytotoxic T lymphocyte (CTL) activity. Radiation therapy (RT) is the main treatment modality used for many types of tu…

Enhancer, chromatin insulator, non-coding RNA and α-histone gene expression during embryogenesis of the sea urchin Paracentrotus lividus.

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the s…

Histone-mediated transgenerational epigenetics

Abstract Epigenetic mechanisms operate at the interface between the environment and genome, by converting the environmental stimuli to phenotypic responses through changes in the chromatin landscape, which ultimately affects gene expression in the absence of alterations in DNA sequence. In this scenario, transgenerational inheritance occurs when epigenetic variations induced by environmental stimuli are transmitted through the germ line to succeeding generations that had never experienced those stimuli. There is an ever-growing list of reports indicating that histones are fundamental players in these processes in a variety of organisms. In this chapter, we provide a perspective on histone-d…

A compact cis-regulatory module defines the exclusive expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm

In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. We have explored the cis-regulatory apparatus of the gene and found that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined Aboral Ectoderm Module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for …

Roles of the Core Components of the Mammalian miRISC in Chromatin Biology

The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have …

cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo

AbstractEmbryonic development is coordinated by networks of evolutionary conserved regulatory genes encoding transcription factors and components of cell signalling pathways. In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. To obtain insights on the regulation of Hbox12 expression, we have explored the cis-regulatory apparatus of the gene. In this paper, we show that the intergenic region …

The Expanding Constellation of Histone Post-Translational Modifications in the Epigenetic Landscape

The emergence of a nucleosome-based chromatin structure accompanied the evolutionary transition from prokaryotes to eukaryotes. In this scenario, histones became the heart of the complex and precisely timed coordination between chromatin architecture and functions during adaptive responses to environmental influence by means of epigenetic mechanisms. Notably, such an epigenetic machinery involves an overwhelming number of post-translational modifications at multiple residues of core and linker histones. This review aims to comprehensively describe old and recent evidence in this exciting field of research. In particular, histone post-translational modification establishing/removal mechanism…

Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway

Mouse mesoangioblasts are vessel-associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll-like receptor …

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl…

Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Ou…

CHOMATIN DYNAMIC AND REGULATION OF GENE EXSPRESSION IN THE SEA URCHIN EMBRYO

Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway

Mouse mesoangioblasts are vessel-associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll-like receptor …

EGFR signalling is required for Paracentrotus lividus endomesoderm specification

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-alpha, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axi…

Isolation and characterization of sea urchin P. lividus microbiota from coelomic fluid

The Sea Urchin sns5 Chromatin Insulator Shapes the Chromatin Architecture of a Lentivirus Vector Integrated in the Mammalian Genome.

Lentivirus vectors are presently the favorite vehicles for therapeutic gene transfer in hematopoietic cells. Nonetheless, these vectors integrate randomly throughout the genome, exhibiting variegation of transgene expression due to the spreading of heterochromatin into the vector sequences. Moreover, the cis-regulatory elements harbored by the vector could disturb the proper transcription of resident genes neighboring the integration site. The incorporation of chromatin insulators in flanking position to the transferred unit can alleviate both the above-mentioned dangerous effects, due to the insulator-specific barrier and enhancer-blocking activities. In this study, we report the valuable …

HBOX12 GENE EXPRESSION AND CELL SPECIFICATION

Composition and geographic variation of the bacterial microbiota associated with the coelomic fluid of the sea urchin Paracentrotus lividus

AbstractIn the present work, culture-based and culture-independent investigations were performed to determine the microbiota structure of the coelomic fluid of Mediterranean sea urchin Paracentrotus lividus individuals collected from two distinct geographical sites neighboring a high-density population bay and a nature reserve, respectively. Next Generation Sequencing analysis of 16S rRNA gene (rDNA) showed that members of the Proteobacteria, Bacteroidetes and Fusobacteria phyla, which have been previously reported to be commonly retrieved from marine invertebrates, dominate the overall population of microorganisms colonizing this liquid tissue, with minority bacterial genera exhibiting rem…

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere tran…

The zebrafish facility of the Mediterranean Center for Human Health Advanced Biotechnologies

The Genome of the Sea Urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus , a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Developmental effects of the protein kinase inhibitor kenpaullone on the sea urchin embryo

The selection and validation of bioactive compounds require multiple approaches, including in-depth analyses of their biological activity in a whole-animal context. We exploited the sea urchin embryo in a rapid, medium-scale range screening to test the effects of the small synthetic kinase inhibitor kenpaullone. We show that sea urchin embryos specifically respond to this molecule depending on both dose and timing of administration. Phenotypic effects of kenpaullone are not immediately visible, since this molecule affects neither the fertilization nor the spatial arrangement of blastomeres at early developmental stages. Nevertheless, kenpaullone exposure from the beginning of embryogenesis …

Identification of a putative GAGA factor in P. lividus embryos

Author response: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

The Sea Urchin sns5 Chromatin Insulator Improves the Likelihood of Lentiviral Vectors in Erythroid Milieu By Organizing an Independent Chromatin Domain at the Integration Site

Abstract Retroviral vectors are currently the most suitable vehicles for therapeutic gene transfer in hematopoietic stem cells. However, these vectors are known to integrate rather randomly throughout the genome, suffering the so called chromosomal position effects (PE). Such a critical occurrence most probably depends upon the ability of heterochromatin to spread in the inserted vector sequences. Moreover, the use of transgenes imply genotoxicity effects, since the cis-regulatory sequences harbored by the vector can disturb the proper transcription of the resident genes neighboring the integration site, potentially leading to malignant transformation. Due to their enhancer blocker activity…

Functional Studies of Regulatory Genes in the Sea Urchin Embryo

Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and show how it has been used in gene perturbation analyses and dissection o…

Gene regulatory network of the early sea urchin embryo involve the Hbox12 homeodomain-containing regulator