The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we ...sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.
Transposable elements (TEs) are active in neuronal cells raising the question whether TE insertions contribute to risk of neuropsychiatric disease. While genome‐wide association studies (GWAS) serve ...as a tool to discover genetic loci associated with neuropsychiatric diseases, unfortunately GWAS do not directly detect structural variants such as TEs. To examine the role of TEs in psychiatric and neurologic disease, we evaluated 17,000 polymorphic TEs and find 76 are in linkage disequilibrium with disease haplotypes (P < 10−6) defined by GWAS. From these 76 polymorphic TEs, we identify potentially causal candidates based on having insertions in genomic regions of regulatory chromatin and on having associations with altered gene expression in brain tissues. We show that lead candidate insertions have regulatory effects on gene expression in human neural stem cells altering the activity of a minimal promoter. Taken together, we identify 10 polymorphic TE insertions that are potential candidates on par with other variants for having a causal role in neurologic and psychiatric disorders.
Synopsis
This study reviews 17,000 polymorphic transposable elements and identifies insertions genetically associated with neuropsychiatric disorders. Features of lead examples are on par with other variants for having a causal role in neurologic and psychiatric disorders.
We identify polymorphic transposable elements that are genetically linked to risk of neuropsychiatric disorders.
Lead transposable element insertions have regulatory activity in a promoter assay tested in human neural stem cells.
Polymorphic transposable elements are associated with altered expression of potential disease genes in neurologic tissues.
This study reviews 17,000 polymorphic transposable elements and identifies insertions genetically associated with neuropsychiatric disorders. Features of lead examples are on par with other variants for having a causal role in neurologic and psychiatric disorders.
The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe here an efficient ...transformation procedure for use in the fission yeast Schizosaccharomyces pombe. This method relies on chemical agents (lithium acetate, and polyethylene glycol) and temperature stresses, which ultimately facilitate transfer of the genetic material through the cell wall and plasma membrane without significant impact on the transferred DNA or the recipient cell. Using this protocol, we consistently see transformation efficiencies between 1.0 × 10
and 1.0 × 10
transformants per microgram of the plasmid with 10
S. pombe cells. The principal benefits and advantages of this method are its simplicity, efficiency, and relative speed of completion.
Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the ...selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.
We present an efficient and organized method of lithium acetate and polyethylene glycol-based transformation of plasmid DNA into the commercially available collection of Schizosaccharomyces pombe ...with single-gene deletions. We also describe how to prepare a duplicate collection of the deletion strains in order to preserve the longevity of the master set. These protocols are adapted to the 96-well format of the 3004 strains of the Version 2.0 Bioneer set but can also be used for later releases of the collection. This transformation method typically yields efficiencies in the range between 1.0 × 10
and 1.0 × 10
transformants per microgram of plasmid DNA. However, some deletion strains transformed with significantly lower efficiencies. We provide a list of these difficult-to-transform strains. Applications for this methodology include the transformation of the deletion set with plasmids necessary for genetic screens.
The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was ...organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.
Transposon insertion sequencing (TIS) is a highly effective method used with bacteria to identify genes important for growth in any condition of interest. Previously, we adapted this method to ...identify essential genes of the yeast Schizosaccharomyces pombe. Here, we describe modifications used to identify genes necessary for the formation of centromeric heterochromatin.
For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).
Display omitted
•Procedure to prepare strains with plasmids that produce Hermes transposition•Method to measure the accumulation of transposition events in liquid cultures•Selection strategy to identify genes important for heterochromatin formation•Library preparation and computational pipeline to analyze insertion site profiles
Transposon insertion sequencing (TIS) is a highly effective method used with bacteria to identify genes important for growth in any condition of interest. Previously, we adapted this method to identify essential genes of the yeast Schizosaccharomyces pombe. Here, we describe modifications used to identify genes necessary for the formation of centromeric heterochromatin.
Retroviruses and long terminal repeat retrotransposons rely on integrase (IN) to insert their complementary DNA (cDNA) into the genome of host cells. Nevertheless, in the absence of IN, retroelements ...can retain notable levels of insertion activity. We have characterized the IN-independent pathway of Tf1 and found that insertion sites had homology to the primers of reverse transcription, which form single-stranded DNAs at the termini of the cDNA. In the absence of IN activity, a similar bias was observed with HIV-1. Our studies showed that the Tf1 insertions result from single-strand annealing, a noncanonical form of homologous recombination mediated by Rad52. By expanding our analysis of insertions to include repeat sequences, we found most formed tandem elements by inserting at preexisting copies of a related transposable element. Unexpectedly, we found that wild-type Tf1 uses the IN-independent pathway as an alternative mode of insertion.
Transposable elements and their remnants constitute a substantial fraction of eukaryotic genomes. Host genomes have evolved defence mechanisms, including chromatin modifications and RNA interference, ...to regulate transposable elements. Here we describe a genome surveillance mechanism for retrotransposons by transposase-derived centromeric protein CENP-B homologues of the fission yeast Schizosaccharomyces pombe. CENP-B homologues of S. pombe localize at and recruit histone deacetylases to silence Tf2 retrotransposons. CENP-Bs also repress solo long terminal repeats (LTRs) and LTR-associated genes. Tf2 elements are clustered into 'Tf' bodies, the organization of which depends on CENP-Bs that display discrete nuclear structures. Furthermore, CENP-Bs prevent an 'extinct' Tf1 retrotransposon from re-entering the host genome by blocking its recombination with extant Tf2, and silence and immobilize a Tf1 integrant that becomes sequestered into Tf bodies. Our results reveal a probable ancient retrotransposon surveillance pathway important for host genome integrity, and highlight potential conflicts between DNA transposons and retrotransposons, major transposable elements believed to have greatly moulded the evolution of genomes.
Fitness Landscape of the Fission Yeast Genome Grech, Leanne; Jeffares, Daniel C; Sadée, Christoph Y ...
Molecular biology and evolution,
08/2019, Letnik:
36, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, ...particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3'- and 5'-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.
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