Further Delineation of Duplications of ARX Locus Detected in Male Patients with Varying Degrees of Intellectual Disability

The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, pae-diatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telen-cephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that—depending on the involvement of tissue-specific enhancers—the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance

Roles of transposable elements in the different layers of gene expression regulation

The biology of transposable elements (TEs) is a fascinating and complex field of investigation. TEs represent a substantial fraction of many eukaryotic genomes and can influence many aspects of DNA function that range from the evolution of genetic information to duplication, stability, and gene expression. Their ability to move inside the genome has been largely recognized as a double-edged sword, as both useful and deleterious effects can result. A fundamental role has been played by the evolution of the molecular processes needed to properly control the expression of TEs. Today, we are far removed from the original reductive vision of TEs as “junk DNA”, and are more convinced that TEs represent an essential element in the regulation of gene expression. In this review, we summarize some of the more recent findings, mainly in the animal kingdom, concerning the active roles that TEs play at every level of gene expression regulation, including chromatin modification, splicing, and protein translation

Cadmium effects on protamine-like and on DNA oxidative damage in Mytilus galloprovincialis

Heavy metal pollution has become one of the most serious environmental problems. They can make DNA damage, due to their ability to form reactive oxygen species, causing mutations in human cells which may cause tumors. In order to monitor sea levels of heavy metals are used specific bioindicators like Mytilus galloprovincialis whose sperm chromatin is organized by three protamine-like (PLII, PLIII and PLIV), which belong to sperm nuclear basic proteins. We report the effects of Cd2+ on DNA, PLII and PLIII. Both PLII and PLIII acquired a slight proteinase K digestion resistance in the presence of metal. We also analyzed the self-association ability of PLII and PLIII Cd2+mediated. Turbidity assay showed that cadmium caused PLII but not PLIII aggregation, while glutaraldehyde produced a reduction of self-association ability for both proteins. Further, Cd2+ induced a decreasing of PLII DNA binding affinity but an increasing for PLIII and promoted H2O2 DNA damage even in the presence of Protamine-like. Those effects may result in an incorrect chromatin packaging that could decrease Mytilus galloprovincialis’ fitness and that of other marine species exposed to this polluting agent

Studies on heavy metals effects on Mytilus galloprovincialis sperm chromatin proteins and on DNA

Heavy metal pollution has become one of the most serious environmental problems in the world that brought threat to human health and ecological security. To monitor sea levels of heavy metals we used as specific bioindicator Mytilus galloprovincialis whose sperm chromatin is organized mainly by three protamine-like proteins (PLII, PLIII and PLIV). These proteins protect DNA from a variety of potentially dangerous reactive species but chromatin packaging doesn’t protect DNA from metal ion-dependent damage in the presence of hydroxyl radicals. We report the effects induced by Cu2+, Ni2+ and Cd2 ions on DNA and on Mytilus galloprovincialis’ PLII and PLIII. Self-association ability of PLII and PLIII mediated by Cd2+, Ni2+ and Cu2+ show that Cd2+ causes only PLII aggregation to a greater extend respect to Ni2+ that induces a mild aggregation of both PLII and PLIII; Cu2+, instead, doesn’t produce any type of aggregates. In vivo studies show that these heavy metals affect PLII, PLIII and PLIV state and properties. While nickel produces an increase of DNA binding affinity for both PLII and PLIII cadmium causes a decrease of PLII DNA binding affinity but an increase for PLIII. Copper and cadmium promote hydrogen peroxide damage of DNA also in the presence of protamine-like proteins while nickel doesn’t. All these effects can lead an anomalous chromatin packaging and a reduction of the reproductive fitness in Mytilus galloprovincialis and other organisms exposed to the same toxicants

Effects of copper and nickel on protamine like and on DNA oxidative damage in Mytilus galloprovincialis

In sperm cells, DNA is complexed with sperm nuclear basic proteins (SNBPs) to form chromatin. Although SNBPs protect DNA from a variety of potentially dangerous reactive species, such as hydroxyl radicals (•OH), chromatin packaging doesn’t protect DNA from metal ion-dependent damage in the presence of hydroxyl radicals. We report the effects induced by Cu2+ and Ni2+ ions on DNA and Mytilus galloprovincialis’ protamine-like II and III. Protamine-like III acquires proteinase k digestion resistance in the presence of copper and not in the presence of nickel; PLII, in contrast with PLIII, is sensitive to proteinase k digestion in presence of both metals. Further, copper promotes hydrogen peroxide damage of DNA also in the presence of PL-types while nickel promoting PL-types aggregation enhances DNA compactness and protection. Nickel in fact, in contrast to copper, increases PLII and PLIII DNA binding affinity. All these effects can lead an anomalous chromatin packaging and, therefore, a reduction of Mytilus galloprovincialis’ reproductive fitness and of other organisms exposed to the same toxicants

New insights into protamine-like component organization in Mytilus galloprovincialis sperm chromatin

We have analyzed Mytilus galloprovincialis’ sperm chromatin which consists of three sperm nuclear basic proteins of the PL-type: PL-II, PL-III and PL-IV, in addition to a residual amount of the four core histones. The knowledge of Mytilus galloprovincialis’ sperm chromatin structure, the organization of the PL-type component in chromatin and their interactions with DNA is of considerable interest because these organisms are largely utilized as bioindicator of marine pollution that could affect its state. Our results obtained by micrococcal nuclease digestion in combination with salt fractionation suggest the existence of a likely unusual organization in which there would be a more accessible location of PL-II/PL-IV when compared to PL-III and core histones. Further we used electrophoretic mobility shift assay in order to define DNA binding mode of PLII and PLIII and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. On the base of our results we propose a model of Mytilus galloprovincialis’ sperm chromatin organization that could be useful in order to develop chromatin-based genotoxicity tests in pollution biomonitoring programs

Antibacterial, antifungal and antitumor activity of Mytilus galloprovincialis’ protamine-like

We have studied the antibacterial, antifungal and antitumor activity of Mytilus galloprovincialis’ protamine-like. Nine bacterial strains were employed. They included two Gram-positive and seven Gram-negative bacteria. The same clinically isolated (CI) bacterial strains were used to compare the sensitivity to these proteins. Antifungal tests were done on 3 strains of fungi including: 1 potentially pathogenic yeast (Candida albicans CI) and 2 filamentous phytopathogenic fungi Botrytis cinerea and Rhyzoctonia solani. Mytilus galloprovincialis’ protamine-like inhibited all bacteria strains with different MBC values ranging from 7.8 to 250 µg/mL and resulted active also on some tested bacteria strains that are generally resistant to conventional antibiotics. They showed also a fungistatic effect on all the fungi tested, with MFCs ranging from 16 to 32 µg/mL and cytotoxic effects on RKO colon cancer cells starting from a concentration of protamine-like 15 µg/mL