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…

Terapia genica delle emoglobinopatie:stato dell'arte

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…

Additional file 4: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Supplementary Results section. (PDF 123 kb)

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

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissoc…

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

Impact of the inclusion of hydrotalcite on the morphological and electrical characteristics of an epoxy-based CNT nanocomposite

The experimental results concerning the correlation between morphological and electrical characteristics of a MWCNT/epoxy based composites are presented. The impact of a nanoclay filler is investigated on the DC conductivity of a matrix loaded with different concentrations of MWCNT in order to evaluate the improvements on the dispersion of CNT inside the epoxy matrix. The dispersion is deemed to be very influencing on the electrical conductivity of the nanocomposite. A detailed morphological and structural characterization allows to interpret the electrical behaviour of the nanocomposites. © 2012 IEEE.

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…

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…

p53-Mediated downregulation of H ferritin promoter transcriptional efficiency via NF-Y.

The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phe…

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…

Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024.

Background The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. R…

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 …

Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3′ end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A…

Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos: The blockage of constitutive hsp70 impairs mitosis

Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividus is shown by means of in situ immunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 …

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…

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…

Sea Urchin sns chromatin insulator prevents silencing and position effect variagation of oncoretroviral vectors transgene expression in murine cell line

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…

Sea urchin HSF activity in vitro and in transgenic embryos.

Evidence is provided for the presence at the physiological temperature of 20 degrees C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividus embryos. This HSF is able to specifically bind in vitro the heat shock element, HSE, of the promoter of the hsp70 gene i.v., as suggested by DNA-protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31 degrees C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivo the transcription of the beta-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea …

EPIGENETIC OF SILENCING OF PARACENTROTUS LIVIDUS ALFA-H2A GENE

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…

The Sea Urchin sns Insulator Blocks CMV Enhancer following Integration in Human Cells

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter up-stream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, p…

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…

Additional file 5: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Figure S1-S6 with corresponding figure legends. (PDF 511 kb)

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…

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-…

Characterization of a nuclear factor associated to the chromatin of sea urchin histone genes

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

DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) alpha-tubulin genes.

To study the molecular aspects of the regulation of transcription of a multigene family, we have isolated and sequenced cDNA and genomic clones coding for the alpha-tubulin of the sea urchin Paracentrotus lividus. Two cDNA clones, P alpha 10 and P alpha 4, contain respectively the coding information for 391 C-terminal and for 338 N-terminal amino acids of the 452 residues that constitute the complete protein. They show silent nucleotide substitutions only, suggesting that P alpha 10 and P alpha 4 represent the cloned copies of two allelic gene transcripts, which encode for two alpha-tubulin isoforms with identical amino acid sequence in the region of the overlap. The comparison of the predi…

Functional characterization and use in the retrovirus vectors of the sea urchin SNS chromatin insulator in erythroid cells

Additional file 5: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Figure S1-S6 with corresponding figure legends. (PDF 511 kb)

The Sea Urchin sns5 Insulator Protects Retroviral Vectors From Chromosomal Position Effects by Maintaining Active Chromatin Structure

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chr…

321. Sea Urchin sns Chromatin Insulator Prevents Silencing and Positional Effect Variegation of Oncoretroviral Vectors Transgene Expression in Murine Erythroid Cell Line

Silencing and position effect are considered significant obstacles to obtain a consistent level of transgene expression in viral gene therapy. Furthermore recent studies had shown that retroviruses tend to land on active genes with the potential consequence of insertional mutagenesis. The inclusion of elements, such as chromatin insulators, capable to insulate a gene from the surrounding chromatin effects at the integration site should improve both efficacy and safety of gene therapy vectors. We have previously characterized a 265 bp insulator element, termed sns, localized at the 3’ end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-…

Spatially restricted expression of PlOtp, a Paracentrotus lividus Orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos

ABSTRACT Several homeobox genes are expressed in the sea urchin embryo but their roles in development have yet to be elucidated. Of particular interest are homologues of homeobox genes that in mouse and Drosophila are involved in patterning the developing central nervous system (CNS). Here, we report the cloning of an orthopedia (Otp)-related gene from Paracentrotus lividus, PlOtp. Otp is a single copy zygotic gene that presents a unique and highly restricted expression pattern. Transcripts were first detected at the mid-gastrula stage in two pairs of oral ectoderm cells located in a ventrolateral position, overlying primary mesenchyme cell (PMC) clusters. Increases in both transcript abund…

Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection

Hepatitis B virus deoxyribonucleic acid (HBV-DNA) was studied by Southern blot analysis in liver biopsy specimens from 75 HBsAg-positive patients with chronic liver disease living in southern Italy. Twenty-seven of the patients were hepatitis delta virus (HDV) superinfected. Intrahepatic HBV-DNA was detected in 54 (72%) patients, 32 (59%) of them with replicative forms. The presence of replicative forms was directly related to liver HBcAg and inversely related to liver HDAg, as shown by multivariate analysis. However, 14 patients with intrahepatic HBV-DNA non-replicative pattern and about half of HDV-infected patients were liver HBcAg and/or serum HBV-DNA positive, mostly in low amounts. Hi…

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…

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

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.

Abstract Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3′ end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communi…

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…

Additional file 4: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Supplementary Results section. (PDF 123 kb)

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…

Gene expression during early embryogenesis of sea urchin: The histone and homeobox genes

Transcriptional regulators are thought to play a key role in cell fate determination and territorial specification in sea urchin. Our goals are to clone transcription factors for studying embryonic development. One approach has been to use promoter binding and gene transfer technology to investigate the mechanisms of transcriptional activation and repression of the early H2A histone gene. By this analysis we identified a transcriptional activator, the MBF-1, that binds to the modulator element of the H2A gene and enhances the activity of the H2A promoter. However, the enhancer activity of the modulator and its interaction with MBF-1 also occurs at the gastrula stage when the early histone g…

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…

The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene.

Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of “modulator”. The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered. This particular enhancer transactivates transcription of the sea urchin early (α) histone H2A gene which is regulated in early sea urchin development. We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes. We conclude that the H2A enha…

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 …

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 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…

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end

Abstract Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the mod…

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

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…

Expression of homeobox-containing genes in the sea urchin (Parancentrotus lividus) embryo

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin geonomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage a…

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…

Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes.

Micrococcal nuclease (MNase) and DNaseI have made a great contribution to our present understanding of the structural organization of the eucaryotic genome [l - 31. The enhanced sensitivity of active portions of the genome to DNaseI gave, in fact, the first indication of differences in the nucleoproteic arrangement of the transcribed as compared to the silent DNA regions [4]. The use of MNase, as a probe of the chromatin organization of specific genes in the active and inactive state, provided additional evidence for this and further showed that the packing of several coding segments of DNA in a regular array of nucleosomal particles is severely but reversibly affected by the transcriptiona…

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…

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, th…

Identification of a putative GAGA factor in P. lividus embryos

Highly restricted expression at the ectoderm–endoderm boundary of PIHbox 9, a sea urchin homeobox gene related to the human HB9 gene

Abstract Characterisation of a sea urchin (P. lividus) homeobox gene PIHbox 9 is reported. The homeodomain of PIHbox9 is 95% identical to the homeodomain of the human HB9 gene, indicating that the two genes are highly related. Temporal expression analysis during sea urchin embryogenesis showed an absence of transcripts at early cleavage stages. At late gastrula stage, transcripts were barely detectable and reached the highest abundance at prism/early pluteus stages. By whole mount in situ hybridisation we observed a highly restricted expression in a few cells of the ectoderm–endoderm boundary of embryos at the prism stage. At pluteus stages, expression of PIHbox 9 was confined around the an…

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…

Additional file 2: Table S2. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at D substages. (XLS 107 kb)

Additional file 6: Table S5. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, abundance profile and mass spectrometry identification parameters of differentially represented spots containing multiple protein components. (XLS 45 kb)

Additional file 3: Table S3. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from membrane proteome analysis at A substages. (XLSX 37 kb)

Additional file 2: Table S2. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at D substages. (XLS 107 kb)

Additional file 6: Table S5. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, abundance profile and mass spectrometry identification parameters of differentially represented spots containing multiple protein components. (XLS 45 kb)

Additional file 7: Table S4. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented proteins due to NAI-107 exposure in Microbispora ATCC-PTA-5024 RP0 strain. (XLSX 32 kb)

Additional file 1: Table S1. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at A substages. (XLSX 48 kb)

Additional file 8: Table S6. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Numbers of KEGG orthology groups participating in molecular and metabolic processes as inferred from genome and proteome analyses, respectively. (XLS 24 kb)

Additional file 3: Table S3. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from membrane proteome analysis at A substages. (XLSX 37 kb)

Additional file 7: Table S4. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented proteins due to NAI-107 exposure in Microbispora ATCC-PTA-5024 RP0 strain. (XLSX 32 kb)

Additional file 8: Table S6. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Numbers of KEGG orthology groups participating in molecular and metabolic processes as inferred from genome and proteome analyses, respectively. (XLS 24 kb)

Additional file 1: Table S1. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at A substages. (XLSX 48 kb)