Transcription Analysis of yZSJ025
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE219048 | GEO | 2022/12/30
REPOSITORIES: GEO
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Project description:We designed and synthesized synI, which is ~21.4% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance, and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. We constructed additional fusion chromosomes to investigate effects of fusions on nuclear function. We observed unexpected loops and twisted structures in chrIII-I and chrIX-III-I fusion chromosomes dependent on silencing protein Sir3. ChrI faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions engineered into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of meiotic recombination protein Red1. These effects extended over >100kb, to disproportionally promote meiotic recombination of small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.
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Project description:A chromosome size-dependent bias in meiotic recombination is in place to ensure that homologous chromosome pairing occurs for all chromosomes, including the smallest ones. This bias is clearly detectable during the assembly of the meiotic protein axis, the structure that compacts meiotic chromosomes and promotes recombination.To investigate the origin of the size bias, we mapped genome-wide occupancy of the meiotic axis protein Red1 in yeast strains containing chromosome fusions and synthetic chromosomes. Meiosis studies of the fusion and synthetic chromosomes further revealed that core centromeres influence the deposition of the axial element protein Red1 over distances >100kb, while pericentromeric regions co-evolved to reduce Red1 binding near centromeres and spread out Red1 along the chromosomes.
2021-05-21 | GSE114731 | GEO
Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking. RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq
2016-09-02 | E-GEOD-81351 | biostudies-arrayexpress
Project description:Deep sequencing has the potential to provide great insight into the composition of the antibody repertoire and is now widely used. To date there has not been a study directly comparing relative rearrangement frequencies obtained with unbiased amplification from genomic DNA (gDNA) and cDNA from the same set of cells to elucidate any differences. Here we use a recently developed method for unbiased amplification of gDNA and directly compare the data to the unbiased RNA IgK repertoire from the same pre-B cell pool. We find that ~20% of Vk genes have relative rearrangement frequencies > 2-fold up or down in the RNA vs DNA libraries, including many members of the Vk4, Vk3 and Vk6 families. Regression analysis indicates that Ikaros and E2A binding are associated with strong promoters. Within the pre-B repertoire, we found that the vastly unequal Vk gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vk genes in pre-B cells are most predictive of rearrangement levels. Comparison of IgK rearrangements that occur in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias towards usage of Jk-distal Vk genes, particularly Vk 10-96 and surrounding genes. Regression analysis indicates that PU.1 binding is the highest predictor of high Vk gene rearrangement frequency in pro-B cells. Lastly, repertoires of iEk -/- pre-B cells reveal that the intronic enhancer actively influences Vk gene usage and displays a germline transcriptional sphere of influence extending to Vk 6-15, overlapping with the deficit in Jk proximal Vk gene usage. These represent previously unknown roles for iEk in addition to its major function in stimulating overall IgK rearrangement.
2018-10-01 | GSE113306 | GEO
Project description:Meiotic crossover (CO) recombination is a process in which programmed DNA double-strand breaks (DSBs) around hotspots are repaired by homologous chromosomes (homologs), and thus exchanges DNA molecules between two homologs. CO is critical for most sexually-reproducing organisms because it promotes accurate chromosome segregation and creates genetic diversity. Therefore, faithful accomplishment of CO formation must be ensured, but the bases of the regulation are not fully understood. Our previous study using fission yeast has revealed that mutants lacking the conserved histone H2A.Z are defective in DSB formation but maintain CO frequency at three loci tested. Here, we tested five additional sites to show that H2A.Z-lacking cells exhibit normal and increased CO frequency at two and three loci, respectively. Detailed interrogation on one of the intervals revealed that CO increase in the mutant would be genetically separable from DSB reduction, and regulated at least partly at the recombination intermediate level. In addition, through genetic analyses of model regions containing a hotspot flanked by two markers as well as genome-wide analyses, we propose that, in the absence of H2A.Z, CO arose from non-hotspot DSBs may compensate for DSB reduction at hotspots. These observations provide clues to further our understanding on CO control.
2021-12-21 | GSE124293 | GEO
Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking.
2016-09-02 | GSE81351 | GEO
Project description:Oxidative stress is a common factor threatening genomic stability in almost all aerobic organisms. Using a yeast screening system, we measured the frequency of mitotic recombination was greatly elevated after H2O2 treatment. H2O2 was able to break chromatid directly in G1 synchronized cells and homologous recombination was induced to repair DNA double stand breaks at S/G2 phase. By whole genome SNP microarray and sequencing, the patterns of H2O2 induced loss of heterozygosity (LOH; gene conversion and crossover), chromosomal rearrangement, and aneuploidy changes were revealed. LOH events were the most common genomic alterations induced by H2O2 and were randomly distributed throughout the genome.
2017-11-21 | GSE107178 | GEO
Project description:Oxidative stress is a common factor threating genomic stability in almost all aerobic organisms. Using a yeast screening system, we measured the frequency of mitotic recombination was greatly elevated after H2O2 treatment. H2O2 was able to break chromatid directly in G1 synchronized cells and homologous recombination was induced to repair DNA double stand breaks at S/G2 phase. By whole genome SNP microarray and sequencing, the patterns of H2O2 induced loss of heterozygosity (LOH; gene conversion and crossover), chromosomal rearrangement, and aneuploidy changes were revealed. LOH events were the most common genomic alterations induced by H2O2 and were randomly distributed throughout the genome.
2017-11-15 | GSE106816 | GEO
Project description:In eukaryotes, meiotic recombination events are distributed non-randomly in the genome with certain regions having high levels of recombination (hotspots) and others having low levels (coldspots). Species with similar DNA sequences (for example, chimpanzees and humans) can have strikingly different patterns of hotspots and coldspots. Below, using a microarray analysis that allows us to measure the frequency of the meiosis-specific double-strand DNA breaks (DSBs) of all 6000 yeast genes, we show that mutation of a single gene (SIR2), which encodes a histone deacetylase, significantly changes DSB frequencies of 12% of yeast genes, elevating DSBs of 5% and reducing DSBs of 7%. Many of the genes with repressed recombination are located in large (50-100 kb) regions located near, but not at, the telomeres. Some of the genes with altered frequencies of DSBs (including the ribosomal RNA gene cluster) are known targets of Sir2p deacetylation in the wild-type strain. Keywords: ChIP-chip
2007-02-01 | GSE6245 | GEO
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
2013-08-05 | E-GEOD-47766 | biostudies-arrayexpress