Distant Genomic Regions Research Articles

Diversification of Antibodies: From V(D)J Recombination to Somatic Exon Shuffling.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

The dynamics of partition protein B (ParB) spreading on the DNA and bridging-induced DNA condensation.

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ∼15 kb away from the loading site and form a partition complex. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins as well as its mechanism in DNA condensation. Using in vitro single-molecule fluorescence imaging, we here show that ParB proteins can load onto the DNA distantly of parS loading site by a novel ParB-ParB recruitment. We observe the proteins loading in-cis and in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. Moreover, we investigate the detailed mechanism of ParB-induced DNA-condensation, where previously spread ParB proteins can fix distant genomic regions in close proximity via transient loop formation. We investigate this process in the light of polymer-polymer phase-separation (PPPS) and detect which molecular domains are involved in this bridging-induced condensation. ParB-ParB recruitment and ParB-DNA condensation explains how ParB can form a bacterial segregation apparatus involving substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell: Part 2-Computational Analysis of Epigenomics Data.

The final data-generation step of genome-wide profiling of any epigenetic parameter typically involves DNA deep sequencing which yields large datasets that must then be computationally analyzed both individually and collectively to comprehensively describe the epigenetic programming that dictates cell fate and function. Here, we describe computational pipelines for analysis of bulk mepigenomic profiling data, including whole-genome bisulfite sequencing (WGBS) to detect DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to detect genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to detect genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to detect 3-dimensional interactions among distant genomic regions. In addition, we describe Chromatin State Discovery and Characterization (ChromHMM) methodology to integrate data from these individual analyses, plus that from RNA-seq analysis of gene expression, to obtain the most comprehensive overall assessment of epigenetic programming associated with gene expression.

Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell-Part 1: Epigenomics Assays.

Epigenomics encompasses analyses of a variety of different epigenetic parameters which, collectively, make up the epigenetic programming that dictates cell fate and function. Here, protocols are provided for four different epigenomic methods including whole-genome bisulfite sequencing (WGBS) to assess DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to assess genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to assess genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to assess three-dimensional interactions among distant genomic regions, plus computational methodology to integrate data from those four methodologies using Chromatin State Discovery and Characterization (ChromHMM) to obtain the most comprehensive overall assessment of epigenetic programming.

Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression.

The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active/inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies. We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method, is opening new avenues to understand how self-assembly of phase-separated nuclear bodies possibly contributes to mammalian genome organization and gene expression.

SvABA: genome-wide detection of structural variants and indels by local assembly.

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20–300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50–300 bp) SVs.

Denise Barlow (1950–2017)—pioneer of genomic imprinting

Life is a fleeting fortune that passes leaving the rest of us transformed by memory. The scientific community is deeply saddened by the loss of Denise Barlow, who was a discoverer in mammalian genetics and gene regulation. Denise passed away peacefully in the presence of friends on October 21, 2017 and leaves behind a legacy of science and researchers inspired by her thinking. Denise studied biology at Reading University and obtained her PhD in Derek Burkes's laboratory at Warwick University in the UK. This was where her interest in developmental biology and especially that of the mammalian embryo arose. After studies of the interferon response, which led her to show that this important viral defense is first observed late in embryogenesis, she moved to London to work with Brigit Hogan at the Imperial Cancer Research Fund (ICRF) laboratories. She followed her desire to study gene expression in the early mouse embryo at a time when the genome sequences of mammals had not yet …

Multiplex Pyrosequencing®: Simultaneous Genotyping Based on SNPs from Distant Genomic Regions.

Pyrosequencing(®) is a technique that allows the quick sequencing of short stretches of DNA, and results can be inspected in real time as they are generated. As such, it is a highly useful tool for the typing of organisms, based on single-nucleotide polymorphisms (SNPs). However, if a single polymorphism is not sufficient for typing and several distant genomic regions need to be examined, multiple Pyrosequencing reactions have to be performed for each organism. This strongly increases both workload and reagent costs. Alternatively, multiplex Pyrosequencing can be performed, in which the multiple sequencing reactions for all analyzed genomic regions are performed in a single reaction. However, when using this method, special care has to be taken while designing the assay and analyzing the results.Here we present a detailed protocol using our newly developed software mPSQed for assay design and MultiPSQ for data analysis.

Abstract 639: Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions.

Abstract It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR). To study DNA DSB repair in vivo, we generated a vector with the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk was the recognition site for the meganuclease I-SceI. The EF1aTk vector was introduced into U937 cells and a clone (designated F5) with a single copy of the vector was isolated. Transfection of F5 cells with an I-SceI expression vector, followed by ganciclovir (GCV) selection, identified clones that lost expression of HsTK. We recovered no GCR, as most of the GCV-resistant clones had deletions of the EF1a promoter or the HsTK. However ∼10% of the clones had insertions (47-756 bp) of DNA sequences derived from distant regions of the genome; all inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE or SINE sequences. Therefore, we hypothesized that the inserted sequences used to “patch” the DNA DSB could be based on reverse transcription of an RNA template. Since sequences derived from human RNA and human genomic DNA are identical (with the exception of RNA splice events, poly-A tails, and RNA-edited nucleotides), we co-transfected the F5 cells with murine RNA and an I-SceI expression vector to determine if patches at the DNA DSB sites could be derived from RNA. We identified 121 independent sequences which represented insertions of 20-609 bp at the I-SceI cleavage site. Of these 121 insertions, 6 were vector or E. coli capture events and 7 were too short to identify. The chromosomal origin of 5 insertions could not be assigned as these insertions consisted of 5 or 6 bp tandem repeat sequences; 3 had a (CCCTAA)n repeat, identical to a mammalian telomere repeat. Of the remaining 103 insertions, 94 were derived from a single genomic loci, and 9 samples contained sequences from more than 2 distinct genomic regions. The sequences were derived from 22 of the 24 human chromosomes. 65% of the sequences were found to contain sequences from a transcribed gene region, and 52% contained repeat sequences such as LINE, SINE, or LTR. The involvement of retrotransposon sequences, which are known to be reverse transcribed and integrated into the genome, supports the hypothesis that at least some of the insertions may be templated from RNA. Although most insertions were derived from human sequences; 9 insertions matched murine sequences, and were derived from the co-transfected mouse RNA. All of these were derived from gene and/or retrotransposon sequence. Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome, and suggest that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Citation Format: Masahiro Onozawa, Tamas Varga, Zhenhua Zhang, Yoo Jung Kim, Peter Aplan. Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 639. doi:10.1158/1538-7445.AM2013-639

Expression Quantitative Trait Loci: Replication, Tissue- and Sex-Specificity in Mice

By treating the transcript abundance as a quantitative trait, gene expression can be mapped to local or distant genomic regions relative to the gene encoding the transcript. Local expression quantitative trait loci (eQTL) generally act in cis (that is, control the expression of only the contiguous structural gene), whereas distal eQTL act in trans. Distal eQTL are more difficult to identify with certainty due to the fact that significant thresholds are very high since all regions of the genome must be tested, and confounding factors such as batch effects can produce false positives. Here, we compare findings from two large genetic crosses between mouse strains C3H/HeJ and C57BL/6J to evaluate the reliability of distal eQTL detection, including "hotspots" influencing the expression of multiple genes in trans. We found that >63% of local eQTL and >18% of distal eQTL were replicable at a threshold of LOD > 4.3 between crosses and 76% of local and >24% of distal eQTL at a threshold of LOD > 6. Additionally, at LOD > 4.3 four tissues studied (adipose, brain, liver, and muscle) exhibited >50% preservation of local eQTL and >17% preservation of distal eQTL. We observed replicated distal eQTL hotspots between the crosses on chromosomes 9 and 17. Finally, >69% of local eQTL and >10% of distal eQTL were preserved in most tissues between sexes. We conclude that most local eQTL are highly replicable between mouse crosses, tissues, and sex as compared to distal eQTL, which exhibited modest replicability.

Condensin and cohesin complexity: the expanding repertoire of functions

Condensin and cohesin complexes act in diverse nuclear processes in addition to their widely known roles in chromosome compaction and sister chromatid cohesion. Recent work has elucidated the contribution of condensin and cohesin to interphase genome organization, control of gene expression, metazoan development and meiosis. Despite these wide-ranging functions, several themes have come to light: both complexes establish higher-order chromosome structure by inhibiting or promoting interactions between distant genomic regions, both complexes influence the chromosomal association of other proteins, and both complexes achieve functional specialization by swapping homologous subunits. Emerging data are expanding the range of processes in which condensin and cohesin are known to participate and are enhancing our knowledge of how chromosome architecture is regulated to influence numerous cellular functions.

Inhibition of Mammary Carcinoma Cell Growth by RXR is Mediated by the Receptor's Oligomeric Switch

Ligands that activate the nuclear receptor retinoid X receptor (RXR) display potent anticarcinogenic activities, but the mechanisms by which these compounds inhibit carcinoma cell growth are poorly understood. While RXR can regulate gene expression due to its intrinsic ligand-activated transcription function, this receptor can also regulate transcription by functioning as a ligand-controlled DNA architectural factor. It was thus reported that apo-RXR self-associates into tetramers and that each dimer within these tetramers can separately bind to an RXR response element. Hence, DNA binding by RXR tetramers may bring distant genomic regions into close physical proximity. As ligand binding induces the dissociation of RXR tetramers into dimers, it can alter gene expression by modulating the DNA architecture. Here, we show that inhibition of mammary carcinoma cell growth by RXR ligands stems from the ability of these compounds to regulate the oligomeric state of RXR and is independent of the direct intrinsic transcriptional activity of the receptor. The data suggest that compounds that trigger dissociation of RXR tetramers may comprise a novel class of anticarcinogenic agents.

A chromatin insulator mediates transgene homing and very long-range enhancer-promoter communication.

Insulator sequences help to organize the genome into discrete functional regions by preventing inappropriate cross-regulation. This is thought to be mediated in part through associations with other insulators located elsewhere in the genome. Enhancers that normally drive Drosophila even skipped (eve) expression are located closer to the TER94 transcription start site than to that of eve. We discovered that the region between these genes has enhancer-blocking activity, and that this insulator region also mediates homing of P-element transgenes to the eve-TER94 genomic neighborhood. Localization of these activities to within 0.6 kb failed to separate them. Importantly, homed transgenic promoters respond to endogenous eve enhancers from great distances, and this long-range communication depends on the homing/insulator region, which we call Homie. We also find that the eve promoter contributes to long-distance communication. However, even the basal hsp70 promoter can communicate with eve enhancers across distances of several megabases, when the communication is mediated by Homie. These studies show that, while Homie blocks enhancer-promoter communication at short range, it facilitates long-range communication between distant genomic regions, possibly by organizing a large chromosomal loop between endogenous and transgenic Homies.

Combined 3C-ChIP-Cloning (6C) Assay: A Tool to Unravel Protein-Mediated Genome Architecture

INTRODUCTIONProgress in technologies to address long-range chromosomal interactions in vivo has extensively revised concepts about different aspects of transcriptional regulation. These methods allow probing physical proximities between chromatin elements without specifically identifying the protein components that mediate such interactions. Here we describe a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to identify the proteins that bridge distant genomic regions, while simultaneously identifying such physical proximities. This method is also useful for determining if a candidate protein might mediate long-range interactions, both in cis and in trans in the nucleus. We discuss how the 6C technique can be incorporated with other techniques to discover all the chromatin regions in the nucleus that interact with a given gene or chromatin region of interest in a specific protein-dependent manner. Such information allows complete, cell-type-specific mapping of all the chromatin interactions mediated by specific proteins. The 6C assay advances our understanding of the three-dimensional aspects of the higher-order folding of chromatin and provides an important tool to examine the role of specific proteins in nuclear organization. In addition to providing a detailed protocol of the 6C technique, we discuss how this technology can be used by investigators working in the area of chromatin biology, with special interest in chromatin long-range interactions.

Natural transformation in the Ralstonia solanacearum species complex: number and size of DNA that can be transferred

Ralstonia solanacearum is a widely distributed phytopathogenic bacterium that is known to invade more than 200 host species, mainly in tropical areas. Reference strain GMI1000 is naturally transformable at in vitro and also in planta conditions and thus has the ability to acquire free exogenous DNA. We tested the ubiquity and variability of natural transformation in the four phylotypes of this species complex using 55 strains isolated from different hosts and geographical regions. Eighty per cent of strains distributed in all the phylotypes were naturally transformable by plasmids and/or genomic DNA. Transformability can be considered as a ubiquitous physiological trait in the R. solanacearum species complex. Transformation performed with two independent DNA donors showed that multiple integration events occurred simultaneously in two distant genomic regions. We also engineered a fourfold-resistant R. solanacearum GMI1000 mutant RS28 to evaluate the size of DNA exchanged during natural transformation. The results demonstrated that this bacterium was able to exchange large DNA fragments ranging from 30 to 90 kb by DNA replacement. The combination of these findings indicated that the natural transformation mechanism could be the main driving force of genetic diversification of the R. solanacearum species complex.

Genomic Analysis of Recombinant Sabin Clinical Isolates

Recombination in Poliovirus vaccine strains is a very frequent phenomenon. In this report 23 polio/Sabin strains isolated from healthy vaccinees or from VAPP patients after OPV administration, were investigated in order to identify recombination sites from 2C to 3D regions of the poliovirus genome. RT-PCR, followed by Restriction Fragment Length Polymorphism (RFLP) screening analysis were applied in four distant genomic regions (5' UTR, VP1, 2C and 3C-3D) in order to detect any putative recombinant. The detected recombinants were sequenced from 2C to the end of the genome (3' UTR) and the exact recombination sites were determined with computational analysis. Five of the 23 isolated strains were recombinant in one genomic region, two of them in 2C, isolates EP16:S3/S2, EP23:S3/S1, two in 3D isolates EP6:S2/S1, EP12:S2/S1 and one in 3A isolate EP9:S2/Sl. Point mutations were found in strains EP3, EP6, EP9 and EP12. Recombination specific types and sites re-occurrence along with point mutations are discussed concerning the polioviruses evolution.

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I- SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase ( HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.

Chromosomal Rearrangements Induced by DNA Double-Strand Breaks.

It has been suggested that introduction of double-strand DNA breaks into human chromosomes can lead to gross chromosomal rearrangements (GCRs) through improper repair mechanisms, and that these GCRs may be leukemogenic through the loss of tumor suppressor genes or the creation of fusion oncogenes. Using the human histiocytic lymphoma cell line U937, we developed a model system in which we were able to introduce a double-strand DNA break (DSB) in vivo at a predetermined location, and subsequently examine the ensuing chromosomal changes flanking the breakage site. In this model, U937 cells are stably transfected with a construct that contains the 18 bp recognition site for a rare cutting endonuclease, I-SceI, whose recognition sequence is not normally present in the human genome. The I-SceI site is placed between a strong constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker by conferring ganciclovir (GCV) sensitivity to the cells. Following transfection of an I-SceI expression vector, double-strand DNA breakage occurs and abrogatesHSV-tk expression, leading to a dramatic increase in GCV resistant cells. We showed that, even in the abscence of GCV selection, the IsceI enzyme was able to cleave its recognition sequence in at least half of the transfected cells. The predominant type of rearrangement detected following the appearance of a double-strand break was an interstitial deletion. These deletions typically displayed uneven recession of free DNA ends flanking the cleavage site, generally resulting in either very short (<10 bp) or very long (>1000) bp deletions extending into the EF1alpha promoter and homogeneous deletions of 100–1700 bp into the HSV-tk gene. Moreover, in many clones, nucleotide microhomologies of 1–6 basepairs, palindromes, or both were detected at the breakpoint junctions, suggesting repair via a non-homologous end joining (NHEJ) mechanism.. We also showed that in 25 % of the recovered clones the DSB was repaired by formation of direct or inverted repeats derived from the breakpoint junction. Several clones showed similarly complex rearrangements, including short nucleotide insertions, inversions and duplications. Finally, approximately 4.5% (7/150) of the clones demonstrated insertion events that involved integration of nucleotide sequences derived from distant genomic regions, including LINE elements, coding regions of endogenous genes and sequences exhibiting known genomic instability. These insertions may have been derived via reverse transcription of mRNA sequences; alternatively the distant genomic regions may have served as templates for the inserted sequences, or genomic DNA fragments may have been directly incorporated into the I-SceI-induced break. A step-by-step modification of this loss of function selection scheme will provide a platform for studying DNA rearrangements induced by faulty dsDNA repair.

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria.

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA(Tyr) is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.

Trans splicing integrates an exon of 22 nucleotides into the nad5 mRNA in higher plant mitochondria.

The genes coding for NADH dehydrogenase subunit 5 (nad5) in mitochondria of the higher plants Oenothera and Arabidopsis are split into five exons that are located in three distant genomic regions. These encode exons a + b, c and d + e, respectively. Maturation of the mRNAs requires two trans splicing events to integrate exon c of only 22 nucleotides. Both trans splicing reactions involve mitochondrial group II intron sequences that allow base pairings in the interrupted domain IV, demonstrating the flexibility of intron structures. The observation of fragmented intron sequences in plant mitochondria suggests that trans splicing is more widespread than previously assumed. RNA editing by C to U alterations in both Oenothera and Arabidopsis open reading frames improves the evolutionary conservation of the encoded polypeptides. Three C to U RNA editing events were observed in intron sequences.