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 with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts

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

The tandemly repeated sea urchin α-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 α-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. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to upregulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3′ insulator and 5′ regulatory regions, in the repression of the α-H2A gene despite the presence of the MBF-1 activator bound to the enhance

Controlled uptake of PFOA in adult specimens of Paracentrotus lividus and evaluation of gene expression in their gonads and embryos

Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years ago it turned out to be a dangerous pollutant which is now of concern for terrestrial and aquatic environments. Here, we investigated the bioaccumulation of PFOA in the sea urchin Paracentrotus lividus after exposure to different concentrations of the pollutant for 28 days. We observed rapid uptake of PFOA in the coelomic fluid collected weekly during the exposure period and high bioaccumulation in gonads at the end of the experiment. Interestingly, animals were also able to fast depurate when relocated to a clean environment. In addition, to assess the effect of PFOA on sea urchins' physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring by causing significant changes in the expression of some key target genes and the occurrence of developmental anomalies in the embryos

Caffeine boosts Ataluren's readthrough activity

The readthrough of nonsense mutations by small molecules like Ataluren is considered a novel therapeutic approach to overcome the gene defect in several genetic diseases as cystic fibrosis (CF). This pharmacological approach suppresses translation termination at premature termination codons (PTCs readthrough) thus restoring the expression of a functional protein. However, readthrough might be limited by the nonsense-mediated mRNA decay (NMD), a cell process that reduces the amount/level of PTCs containing mRNAs. Here we investigate the combined action of Ataluren and caffeine to enhance the readthrough of PTCs. IB3.1 CF cells with a nonsense mutation were treated with caffeine to attenuate the Nonsense-Mediated mRNA Decay (NMD) activity and thus enhance the stability of the nonsense (ns)-CFTR-mRNA to be targeted by Ataluren. Our results show that NMD attenuation by caffeine enhances mRNA stability and more importantly when combined with Ataluren increase the recovery of the full-length CFTR protein

Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations

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 α-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 of the α-H2A gene at gastrula stage. We found that important negative regulative sequences are located in the 462 bp sns 5 fragment located in the 3′ region. Remarkably, sns 5 contains the sns enhancer blocking element and the most 3′ H2A codons. In addition, we made the striking observation that inhibition of the anti-enhancer activity of sns, by titration of the binding proteins in microinjected embryos, also affected the capability of sns 5 to down-regulate transgene expression at gastrula stage. A further sequence element essential for repression of the H2A gene was identified upstream of the enhancer, in the 5′ region, and contains four GAGA repeats. Altogether these findings suggest that down-regulation of the α-H2A gene occurs by the functional interaction of the 5′ and 3′ cis sequence elements. These results demonstrate the involvement of a genomic insulator in the silencing of gene expression

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase

Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs

Preliminary genetic characterisation of Southern Smooth Snake Coronella girondica (Serpentes, Colubridae) populations in Italy, with some considerations on their alpine distribution

The Southern smooth snake, Coronella girondica, is a small-sized colubrid found in Northwest Africa and Southwest Europe. Mitochondrial DNA-based studies showed that the species can be split into five clades: two from Northwest Africa (one Moroccan and one Tunisian-Algerian) and three from Europe (one in the south-west of the Iberian Peninsula, one in the south-east of Spain and one in the rest of the European range). With regards to Italy, to date, only two samples have been analysed both from the Province of Pisa, Tuscany, pointing at that fact that genetic characterisation of Italian populations is still lacking. Accordingly, we have increased the sampling coverage with 19 new samples from northern and central regions of Italy, including two populations, apparently disconnected from the rest of the known range, and analysed their phylogenetic relationships using a portion of the mitochondrial cytochrome b gene. Our results confirm the general phylogenetic arrangement detected in previous studies; specifically for Italian populations, no variability emerged from the Apennine populations, and a slight differentiation could be shown for the Alpine and subalpine ones. This pattern can be explained assuming past spread and recent isolation of C. girondica relict populations in the Alpine region, likely during the Last Glacial Maximum. Later, during the Holocene, the Italian Alps and the Po Plain went through various climatic variations and high anthropization which may have influenced C. girondica distribution through expansion and contraction processes