Bioinformatic survey of CRISPR loci across 15 Serratia species.

The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) system of prokaryotes is an adaptative immune defense mechanism to protect themselves from invading genetic elements (e.g., phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order of Enterobacterales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotica, and ureilytica. Apart from subtypes I-E and I-F1 which had previously been identified in marcescens, we report that of I-C and the I-E unique locus 1, I-E*, and I-F1 unique locus 1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. The plasmid and/or phage origin of spacers was also established.

Evolutive emergence and divergence of an Ig regulatory node: An environmental sensor getting cues from the aryl hydrocarbon receptor?

One gene, the immunoglobulin heavy chain (IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3' end of the IgH gene locus (3'RR). Animal models have demonstrated an essential role of the 3'RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3'RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3'RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3'RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3'RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2.

New insight of human-IgH 3'regulatory regions in immunoglobulins switch.

BACKGROUND: Several studies in animal models have demonstrated the role of the 3' Regulatory Region (3'RR) in the B cell maturation in mammals. In healthy humans, the concentration of each class of circulating immunoglobulins (Igs) has stable but different levels, due to several control mechanisms that also involve a duplicated version of the 3'RR on the chromosome 14 (chr14). The classes' equilibrium can be altered during infections and in other pathological conditions. MATERIAL AND METHODS: We studied the concentrations of IgA, IgM, IgG classes and IgG subclasses in a cohort of 1235 people having immunoglobulin concentrations within normal range to determine the presence of any correlation between the Igs serum concentrations, age and ratio among Ig classes and IgG subclasses in healthy humans. Furthermore, we assessed the concentrations of IgE and the allelic frequency of 3'RR1 hs1.2 enhancer in a group of 115 subjects with high levels of circulating IgE due to acute exacerbation of allergic asthma and in a control group of 118 healthy subjects. RESULTS: In both children and adult subjects, the concentrations of the four IgG subclasses decreased from IgG1 to IgG4. Furthermore, the 3'RR1 enhancer hs1.2 alleles contribute to the control of the IgG subclasses levels, but it does not affect the IgE levels. CONCLUSION: The 3'RR1 controls IgG and IgE through different mechanisms, only in the IgG case involving the hs1.2 alleles. Thus, considering the IgH constant genes loci on the chromosome 14 and the multiple steps of switch that rearrange the whole region, we found that in humans the classes of Igs are modulated by mechanisms involving a complex interaction and transition between 3'RR1 and 3'RR2, also in physiological conditions.

A high-resolution map of small-scale inversions in the gibbon genome.

Gibbons are the most speciose family of living apes, characterized by a diverse chromosome number and rapid rate of large-scale rearrangements. Here we performed single-cell template strand sequencing (Strand-seq), molecular cytogenetics, and deep in silico analysis of a southern white-cheeked gibbon genome, providing the first comprehensive map of 238 previously hidden small-scale inversions. We determined that more than half are gibbon specific, at least fivefold higher than shown for other primate lineage-specific inversions, with a significantly high number of small heterozygous inversions, suggesting that accelerated evolution of inversions may have played a role in the high sympatric diversity of gibbons. Although the precise mechanisms underlying these inversions are not yet understood, it is clear that segmental duplication-mediated NAHR only accounts for a small fraction of events. Several genomic features, including gene density and repeat (e.g., LINE-1) content, might render these regions more break-prone and susceptible to inversion formation. In the attempt to characterize interspecific variation between southern and northern white-cheeked gibbons, we identify several large assembly errors in the current GGSC Nleu3.0/nomLeu3 reference genome comprising more than 49 megabases of DNA. Finally, we provide a list of 182 candidate genes potentially involved in gibbon diversification and speciation.

Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

The Organization of the Pig T-Cell Receptor γ (TRG) Locus Provides Insights into the Evolutionary Patterns of the TRG Genes across Cetartiodactyla.

The domestic pig (Sus scrofa) is a species representative of the Suina, one of the four suborders within Cetartiodactyla. In this paper, we reported our analysis of the pig TRG locus in comparison with the loci of species representative of the Ruminantia, Tylopoda, and Cetacea suborders. The pig TRG genomic structure reiterates the peculiarity of the organization of Cetartiodactyla loci in TRGC "cassettes", each containing the basic V-J-J-C unit. Eighteen genes arranged in four TRGC cassettes, form the pig TRG locus. All the functional TRG genes were expressed, and the TRGV genes preferentially rearrange with the TRGJ genes within their own cassette, which correlates the diversity of the γ-chain repertoire with the number of cassettes. Among them, the TRGC5, located at the 5' end of the locus, is the only cassette that retains a marked homology with the corresponding TRGC cassettes of all the analyzed species. The preservation of the TRGC5 cassette for such a long evolutionary time presumes a highly specialized function of its genes, which could be essential for the survival of species. Therefore, the maintenance of this cassette in pigs confirms that it is the most evolutionarily ancient within Cetartiodactyla, and it has undergone a process of duplication to give rise to the other TRGC cassettes in the different artiodactyl species in a lineage-specific manner.

The Genomic Organisation of the TRA/TRD Locus Validates the Peculiar Characteristics of Dromedary δ-Chain Expression.

The role of γδ T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., "γδ low species") with a restricted diversity of γδ T cell receptors (TR). Conversely, artiodactyl species (i.e., "γδ high species") account for a high proportion of γδ T cells with large γ and δ chain repertoires. The genomic organisation of the TR γ (TRG) and δ (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for γ and δ chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR α (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary δ chains. Furthermore, the comparison between genomic and expressed sequences reveals that D genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary δ chain antigen binding-site.

A genomic survey of Tc1-mariner transposons in nematodes suggests extensive horizontal transposon transfer events.

The number of reports concerning horizontal transposon transfers (HTT) in metazoan species is considerably increased, alongside with the exponential growth of genomic sequence data However, our understanding of the mechanisms of such phenomenon is still at an early stage. Nematodes constitute an animal phylum successfully adapted to almost every ecosystem and for this reason could potentially contribute to spreading the genetic information through horizontal transfer. To date, few studies describe HTT of nematode retrotransposons. This is due to the lack of annotation of transposable elements in the sequenced nematode genomes, especially DNA transposons, which are acknowledged as the best horizontal travelers among mobile sequences. We have therefore started a survey of DNA transposons and their possible involvement in HTT in sequenced nematode genomes. Here, we describe 83 new Tc1/mariner elements distributed in 17 nematode species. Among them, nine families were possibly horizontally transferred between nematodes and the most diverse animal species, including ants as preferred partner of HTT. The results obtained suggest that HTT events involving nematodes Tc1/mariner elements are not uncommon, and that nematodes could have a possible role as transposon reservoir that, in turn, can be redistributed among animal genomes. Overall, this could be relevant to understand how the inter-species genetic flows shape the landscape of genetic variation of organisms inhabiting specific environmental communities.

Analysis of Four New Enterococcus faecalis Phages and Modeling of a Hyaluronidase Catalytic Domain from Saphexavirus.

Background: Phage therapy (PT), as a method to treat bacterial infections, needs identification of bacteriophages targeting specific pathogenic host. Enterococcus faecalis, a Gram-positive coccus resident in the human gastrointestinal tract, may become pathogenic in hospitalized patients showing acquired resistance to vancomycin and thus representing a possible target for PT. Materials and Methods: We isolated four phages that infect E. faecalis and characterized them by host range screening, transmission electron microscopy, and genome sequencing. We also identified and three-dimensional modeled a new hyaluronidase enzyme. Results: The four phages belong to Siphoviridae family: three Efquatrovirus (namely vB_EfaS_TV51, vB_EfaS_TV54, and vB_EfaS_TV217) and one Saphexavirus (vB_EfaS_TV16). All of them are compatible with lytic cycle. vB_EfaS_TV16 moreover presents a gene encoding for a hyaluronidase enzyme. Conclusions: The identified phages show features suggesting their useful application in PT, particularly the Saphexavirus that may be of enhanced relevance in PT because of its potential biofilm-digestion capability.

The establishment of a fungal consortium in a new winery.

The biodiversity and evolution of fungal communities were monitored over a period of 3 vintages in a new winery. Samples were collected before grape receipt and 3 months after fermentation from 3 different wine related environments (WRE): floor, walls and equipment and analyzed using Illumina Mi-Seq. Genera of mold and filamentous fungi (294), non-enological (10) and wine-associated yeasts (25) were detected on all WREs before the arrival of the first harvest. Among them, genera like Alternaria and Aureobasidium persisted during two vintages. Therefore, these genera are not specific to winery environment and appear to be adapted to natural or anthropic environments due to their ubiquitous character. Some genera like Candida were also detected before the first harvest but only on one WREs, whereas, on the other WREs they were found after the harvest. The ubiquitous character and phenotypic traits of these fungal genera can explain their dynamics. After the first harvest and during 3 vintages the initial consortium was enriched by oenological genera like Starmerella introduced either by harvest or by potential transfers between the different WREs. However, these establishing genera, including Saccharomyces, do not appear to persist due to their low adaptation to the stressful conditions of winery environment.

Concerted variation of the 3' regulatory region of Ig heavy chain and Gm haplotypes across human continental populations.

OBJECTIVES: The 3' regulatory region of the immunoglobulin heavy chain gene (IGH) includes the HS1.2 enhancer displaying length polymorphism with four known variants. The goal of the research was to provide an overview of this variability and of its evolutionary significance across human populations. MATERIALS AND METHODS: We compiled published and original data on HS1.2 polymorphism in 3,100 subjects from 26 human populations. Moreover, we imputed the haplotypic arrangement of the HS1.2 region in the 1000 Genomes Project (1KGP). In this dataset, imputation could also be obtained for the G1m-G3m allotype by virtue of the precise correspondence between serological types and amino acid (and DNA) substitutions in IGHG1 and IGHG3. RESULTS: HS1.2 variant frequencies displayed similar patterns of continental partitioning as those reported in the literature for the physically neighboring IGHG1-IGHG3 system. The 1KGP data revealed that linkage disequilibrium (LD) can explain the spread of joint HS1.2-IGHG1-IGHG3 associations across continents and within continental populations, with stronger LD out of Africa and the features of an evolutionarily stable genomic block with differential expression in lymphoblastoid cell lines. DISCUSSION: Strong population structuring involves at least the entire 70 kb genomic region here considered, due to the tight LD which maintained HS1.2, IGHG1, and IGHG3 in nonrandom arrangements. This might be key to better understand the evolutionary path of the entire genomic region driven by immune response capabilities, during the formation of continental gene pools.

Characterization of CRISPR-Cas Systems in Serratia marcescens Isolated from Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae).

The CRISPR-Cas adaptive immune system has been attracting increasing scientific interest for biological functions and biotechnological applications. Data on the Serratia marcescens system are scarce. Here, we report a comprehensive characterisation of CRISPR-Cas systems identified in S. marcescens strains isolated as secondary symbionts of Rhynchophorus ferrugineus, also known as Red Palm Weevil (RPW), one of the most invasive pests of major cultivated palms. Whole genome sequencing was performed on four strains (S1, S5, S8, and S13), which were isolated from the reproductive apparatus of RPWs. Subtypes I-F and I-E were harboured by S5 and S8, respectively. No CRISPR-Cas system was detected in S1 or S13. Two CRISPR arrays (4 and 51 spacers) were detected in S5 and three arrays (11, 31, and 30 spacers) were detected in S8. The CRISPR-Cas systems were located in the genomic region spanning from ybhR to phnP, as if this were the only region where CRISPR-Cas loci were acquired. This was confirmed by analyzing the S. marcescens complete genomes available in the NCBI database. This region defines a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. This study also supplies the first identification of subtype I-E in S. marcescens.

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.

Correction: MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

In the original version of this Article, the affiliation details for Giovanni Martinelli were incorrectly given as 'Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy' and it should have been given as 'Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy and not Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy.'Furthermore, the original version of this Article contained an error in the spelling of the authors Alberto L'Abbate and Pietro D'Addabbo, an acute accent was used instead of an apostrophe for these authors names.These errors have now been corrected in both the PDF and HTML versions of the Article.

Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction.

The presence of tetraplex structures in the promoter region of the myogenic differentiation 1 gene (MyoD1) was investigated with a specific tetraplex-binding porphyrin (TMPyP4), to test its influence on the expression of MyoD1 itself and downstream-regulated genes during myogenic differentiation. TMPyP4-exposed C2C12 myoblasts, blocking MyoD1 transcription, proliferated reaching confluence and fused forming elongated structures, resembling myotubes, devoid of myosin heavy chain 3 (MHC) expression. Besides lack of MHC, upon MyoD1 inhibition, other myogenic gene expressions were also affected in treated cells, while untreated control cell culture showed normal myotube formation expressing MyoD1, Myog, MRF4, Myf5, and MHC. Unexpectedly, the myomaker (Mymk) gene expression was not affected upon TMPyP4 exposure during C2C12 myogenic differentiation. At the genomic level, the bioinformatic comparison of putative tetraplex sites found that three tetraplexes in MyoD1 and Myog are highly conserved in mammals, while Mymk and MHC did not show any conserved tetraplexes in the analysed regions. Thus, here, we report for the first time that the inhibition of the MyoD1 promoter function, stabilizing the tetraplex region, affects downstream myogenic genes by blocking their expression, while leaving the expression of Mymk unaltered. These results reveal the existence of two distinct pathways: one leading to cell fusion and one guaranteeing correct myotube differentiation.

Association between Psoriasis and haplotypes of the IgH 3' Regulatory Region 1.

The association studies of several immune-diseases with the 3' Regulatory Region 1 (3'RR1) increased interest on the role that the region plays in the immune-regulation. The 3'RR1 is a polymorphic region on human chromosome 14q32, acting as a cis-regulative element on the Immunoglobulin constant-gene locus recently considered as super-enhancer. The human 3'RR1 share large sequences with its paralogous 3'RR2, at high level of similarity. Thus, a focused investigation was necessary to discriminate each one of the duplicated components of the two regions and its specific contribution to the immunologic phenotype. One of the duplicated elements is the hs1.2 enhancer. The 3'RR1 alleles of this enhancer were demonstrated to play a role in autoimmune diseases, including Psoriasis. We sequenced a specific region internal to the 3'RR1 in hs1.2 homozygous subjects, to detect SNPs associated to the main alleles of the enhancer. We identified two alternative nine-SNPs haplotypes strictly linked to the allele *1 and *2 of hs1.2, that could be used as markers to further investigate the region and associations to pathology. Finally, we identified two haplotypes, namely E2A1 and E2A2, that strongly support the hypothesis of a relevant effect of the rs35216181 in the onset of Psoriasis when the *2 allele is present.

Inversion variants in human and primate genomes.

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.

MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

Association between IgH enhancer hs1.2 and type 1 diabetes.

AIM: To investigate the association of alleles of the 3' immunoglobulin heavy-chain regulatory region 1 (3'RR-1) enhancer hs1.2 in patients with type 1 diabetes (T1D). METHODS: Eighty-one patients with T1D [among which 12 had concomitant coeliac disease (CD) and 25 an autoimmune thyroid disease (AITD)] were compared to 248 healthy individuals. All subjects were recruited from the same geographical area. Blood samples were collected from all patients and a nested PCR was performed to amplify the core of the 3'RR-1 and detect the alleles of the hs1.2 enhancer. RESULTS: Allele distribution in healthy individuals was significantly different when compared to that of patients with T1D (p < 0.01). Even greater differences were detected comparing allele distribution of patients with T1D alone versus those with concomitant CD, but not versus those with concomitant AITD. The frequency of *2 allele is increased by 23% in patients with T1D and CD. CONCLUSIONS: The present study establishes that the multiallelic hs1.2 enhancer of the 3'RR-1 is associated with T1D, with higher frequency when there is co-occurrence of CD. This evidence has been previously observed in other immune diseases.

Epigenetic origin of evolutionary novel centromeres.

Most evolutionary new centromeres (ENC) are composed of large arrays of satellite DNA and surrounded by segmental duplications. However, the hypothesis is that ENCs are seeded in an anonymous sequence and only over time have acquired the complexity of "normal" centromeres. Up to now evidence to test this hypothesis was lacking. We recently discovered that the well-known polymorphism of orangutan chromosome 12 was due to the presence of an ENC. We sequenced the genome of an orangutan homozygous for the ENC, and we focused our analysis on the comparison of the ENC domain with respect to its wild type counterpart. No significant variations were found. This finding is the first clear evidence that ENC seedings are epigenetic in nature. The compaction of the ENC domain was found significantly higher than the corresponding WT region and, interestingly, the expression of the only gene embedded in the region was significantly repressed.