cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.13049813.v3
title: A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in <i>Drosophila</i>

description: Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of <i>Drosophila melanogaster</i> as well as to other <i>Drosophila</i> species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against <i>eyes absent</i>. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
DOI: 10.6084/m9.figshare.11449767
title: SILAC-based quantitative proteomic analysis of <i>Drosophila</i> gastrula stage embryos mutant for fibroblast growth factor signalling

description: Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. <i>Drosophila melanogaster</i> is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in <i>Drosophila</i> embryos at the gastrula stage. We present a robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol for labelling proteins in early embryos. For the selection of homozygously mutant embryos at the pre-gastrula stage, we developed an independent genetic marker. Our analyses detected quantitative changes in the global proteome of <i>htl</i> mutant embryos during gastrulation. We identified distinct classes of downregulated and upregulated proteins, and network analyses indicate functionally related groups of proteins in each class. In addition, we identified changes in the abundance of phosphopeptides. In summary, our quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in <i>htl</i> mutant embryos.
DOI: 10.6084/m9.figshare.11449767.v1
title: SILAC-based quantitative proteomic analysis of <i>Drosophila</i> gastrula stage embryos mutant for fibroblast growth factor signalling

description: Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. <i>Drosophila melanogaster</i> is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in <i>Drosophila</i> embryos at the gastrula stage. We present a robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol for labelling proteins in early embryos. For the selection of homozygously mutant embryos at the pre-gastrula stage, we developed an independent genetic marker. Our analyses detected quantitative changes in the global proteome of <i>htl</i> mutant embryos during gastrulation. We identified distinct classes of downregulated and upregulated proteins, and network analyses indicate functionally related groups of proteins in each class. In addition, we identified changes in the abundance of phosphopeptides. In summary, our quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in <i>htl</i> mutant embryos.
DOI: 10.6084/m9.figshare.11365820
title: Correlation of Infinium HumanMethylation450K and MethylationEPIC BeadChip arrays in cartilage

description: DNA methylation of CpG sites is commonly measured using Illumina Infinium BeadChip platforms. The Infinium MethylationEPIC array has replaced the Infinium Methylation450K array. The two arrays use the same technology, with the EPIC array assaying almost double the number of sites than the 450K array. In this study, we compare DNA methylation values of shared CpGs of the same human cartilage samples assayed using both platforms. DNA methylation was measured in 21 human cartilage samples using the both 450K and EPIC arrays. Additional matched 450K and EPIC data in whole tumour and whole blood were downloaded from GEO GSE92580 and GSE86833, respectively. Data were processed using the Bioconductor package Minfi. DNA methylation of six CpG sites was validated for the same 21 cartilage samples by pyrosequencing. In cartilage samples, overall sample correlations of methylation values between arrays were high (Pearson’s <i>r</i> &gt; 0.96). However, 50.5% of CpG sites showed poor correlation (<i>r</i> &lt; 0.2) between arrays. Sites with limited variance and with either very high or very low methylation levels in cartilage exhibited lower correlation values, corroborating prior studies in whole blood. Bisulphite pyrosequencing did not highlight one array as generating more accurate methylation values. For a specific CpG site, the array methylation correlation coefficient differed between cartilage, tumour, and whole blood, reflecting the difference in methylation variance between cell types. Researchers should be cautious when analysing methylation of CpG sites that show low methylation variance within the cell type of interest, regardless of the method used to assay methylation.
DOI: 10.6084/m9.figshare.13049813.v2
title: A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in <i>Drosophila</i>

description: Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of <i>Drosophila melanogaster</i> as well as to other <i>Drosophila</i> species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against <i>eyes absent</i>. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
DOI: 10.6084/m9.figshare.11365820.v1
title: Correlation of Infinium HumanMethylation450K and MethylationEPIC BeadChip arrays in cartilage

description: DNA methylation of CpG sites is commonly measured using Illumina Infinium BeadChip platforms. The Infinium MethylationEPIC array has replaced the Infinium Methylation450K array. The two arrays use the same technology, with the EPIC array assaying almost double the number of sites than the 450K array. In this study, we compare DNA methylation values of shared CpGs of the same human cartilage samples assayed using both platforms. DNA methylation was measured in 21 human cartilage samples using the both 450K and EPIC arrays. Additional matched 450K and EPIC data in whole tumour and whole blood were downloaded from GEO GSE92580 and GSE86833, respectively. Data were processed using the Bioconductor package Minfi. DNA methylation of six CpG sites was validated for the same 21 cartilage samples by pyrosequencing. In cartilage samples, overall sample correlations of methylation values between arrays were high (Pearson’s <i>r</i> &gt; 0.96). However, 50.5% of CpG sites showed poor correlation (<i>r</i> &lt; 0.2) between arrays. Sites with limited variance and with either very high or very low methylation levels in cartilage exhibited lower correlation values, corroborating prior studies in whole blood. Bisulphite pyrosequencing did not highlight one array as generating more accurate methylation values. For a specific CpG site, the array methylation correlation coefficient differed between cartilage, tumour, and whole blood, reflecting the difference in methylation variance between cell types. Researchers should be cautious when analysing methylation of CpG sites that show low methylation variance within the cell type of interest, regardless of the method used to assay methylation.
DOI: 10.6084/m9.figshare.11835744
title: Tissue and cancer-specific expression of DIEXF is epigenetically mediated by an Alu repeat

description: Alu repeats constitute a major fraction of human genome and for a small subset of them a role in gene regulation has been described. The number of studies focused on the functional characterization of particular Alu elements is very limited. Most Alu elements are DNA methylated and then assumed to lie in repressed chromatin domains. We hypothesize that Alu elements with low or variable DNA methylation are candidates for a functional role. In a genome-wide study in normal and cancer tissues, we pinpointed an Alu repeat (AluSq2) with differential methylation located upstream of the promoter region of the <i>DIEXF</i> gene. <i>DIEXF</i> encodes a highly conserved factor essential for the development of zebrafish digestive tract. To characterize the contribution of the Alu element to the regulation of <i>DIEXF</i> we analysed the epigenetic landscapes of the gene promoter and flanking regions in different cell types and cancers. Alternate epigenetic profiles (DNA methylation and histone modifications) of the AluSq2 element were associated with <i>DIEXF</i> transcript diversity as well as protein levels, while the epigenetic profile of the CpG island associated with the <i>DIEXF</i> promoter remained unchanged. These results suggest that AluSq2 might directly contribute to the regulation of <i>DIEXF</i> transcription and protein expression. Moreover, AluSq2 was DNA hypomethylated in different cancer types, pointing out its putative contribution to <i>DIEXF</i> alteration in cancer and its potential as tumoural biomarker.
DOI: 10.6084/m9.figshare.11835744.v1
title: Tissue and cancer-specific expression of DIEXF is epigenetically mediated by an Alu repeat

description: Alu repeats constitute a major fraction of human genome and for a small subset of them a role in gene regulation has been described. The number of studies focused on the functional characterization of particular Alu elements is very limited. Most Alu elements are DNA methylated and then assumed to lie in repressed chromatin domains. We hypothesize that Alu elements with low or variable DNA methylation are candidates for a functional role. In a genome-wide study in normal and cancer tissues, we pinpointed an Alu repeat (AluSq2) with differential methylation located upstream of the promoter region of the <i>DIEXF</i> gene. <i>DIEXF</i> encodes a highly conserved factor essential for the development of zebrafish digestive tract. To characterize the contribution of the Alu element to the regulation of <i>DIEXF</i> we analysed the epigenetic landscapes of the gene promoter and flanking regions in different cell types and cancers. Alternate epigenetic profiles (DNA methylation and histone modifications) of the AluSq2 element were associated with <i>DIEXF</i> transcript diversity as well as protein levels, while the epigenetic profile of the CpG island associated with the <i>DIEXF</i> promoter remained unchanged. These results suggest that AluSq2 might directly contribute to the regulation of <i>DIEXF</i> transcription and protein expression. Moreover, AluSq2 was DNA hypomethylated in different cancer types, pointing out its putative contribution to <i>DIEXF</i> alteration in cancer and its potential as tumoural biomarker.
DOI: 10.6084/m9.figshare.11758719
title: Epigenomics of being bullied: changes in DNA methylation following bullying exposure

description: Bullying among children is ubiquitous and associated with pervasive mental health problems. However, little is known about the biological pathways that change after exposure to bullying. Epigenome-wide changes in DNA methylation in peripheral blood were studied from pre- to post measurement of bullying exposure, in a longitudinal study of the population-based Generation R Study and Avon Longitudinal Study of Parents and Children (combined <i>n </i>= 1,352). Linear mixed-model results were meta-analysed to estimate how DNA methylation changed as a function of exposure to bullying. Sensitivity analyses including co-occurring child characteristics and risks were performed, as well as a Gene Ontology analysis. A candidate follow-up was employed for CpG (cytosine-phosphate-guanine) sites annotated to <i>5-HTT</i> and <i>NR3C1</i>. One site, cg17312179, showed small changes in DNA methylation associated to bullying exposure (<i>b </i>= −2.67e-03, SE = 4.97e-04, <i>p </i>= 7.17e-08). This site is annotated to <i>RAB14</i>, an oncogene related to Golgi apparatus functioning, and its methylation levels decreased for exposed but increased for non-exposed. This result was consistent across sensitivity analyses. Enriched Gene Ontology pathways for differentially methylated sites included cardiac function and neurodevelopmental processes. Top CpG sites tended to have overall low levels of DNA methylation, decreasing in exposed, increasing in non-exposed individuals. There were no gene-wide corrected findings for <i>5-HTT</i> and <i>NR3C1</i>. This is the first study to identify changes in DNA methylation associated with bullying exposure at the epigenome-wide significance level. Consistent with other population-based studies, we do not find evidence for strong associations between bullying exposure and DNA methylation.
DOI: 10.6084/m9.figshare.11758719.v1
title: Epigenomics of being bullied: changes in DNA methylation following bullying exposure

description: Bullying among children is ubiquitous and associated with pervasive mental health problems. However, little is known about the biological pathways that change after exposure to bullying. Epigenome-wide changes in DNA methylation in peripheral blood were studied from pre- to post measurement of bullying exposure, in a longitudinal study of the population-based Generation R Study and Avon Longitudinal Study of Parents and Children (combined <i>n </i>= 1,352). Linear mixed-model results were meta-analysed to estimate how DNA methylation changed as a function of exposure to bullying. Sensitivity analyses including co-occurring child characteristics and risks were performed, as well as a Gene Ontology analysis. A candidate follow-up was employed for CpG (cytosine-phosphate-guanine) sites annotated to <i>5-HTT</i> and <i>NR3C1</i>. One site, cg17312179, showed small changes in DNA methylation associated to bullying exposure (<i>b </i>= −2.67e-03, SE = 4.97e-04, <i>p </i>= 7.17e-08). This site is annotated to <i>RAB14</i>, an oncogene related to Golgi apparatus functioning, and its methylation levels decreased for exposed but increased for non-exposed. This result was consistent across sensitivity analyses. Enriched Gene Ontology pathways for differentially methylated sites included cardiac function and neurodevelopmental processes. Top CpG sites tended to have overall low levels of DNA methylation, decreasing in exposed, increasing in non-exposed individuals. There were no gene-wide corrected findings for <i>5-HTT</i> and <i>NR3C1</i>. This is the first study to identify changes in DNA methylation associated with bullying exposure at the epigenome-wide significance level. Consistent with other population-based studies, we do not find evidence for strong associations between bullying exposure and DNA methylation.
DOI: 10.6084/m9.figshare.11979693
title: Chromatin structure regulates cancer-specific alternative splicing events in primary HPV-related oropharyngeal squamous cell carcinoma

description: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumours. In this study, eleven ASEs (<i>GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331</i>, and <i>ELOVL1)</i> were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. <i>In vitro</i> qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and <i>in silico</i> analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (<i>MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2</i>, and <i>ELOVL1)</i> showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.
DOI: 10.6084/m9.figshare.11979693.v2
title: Chromatin structure regulates cancer-specific alternative splicing events in primary HPV-related oropharyngeal squamous cell carcinoma

description: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumours. In this study, eleven ASEs (<i>GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331</i>, and <i>ELOVL1)</i> were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. <i>In vitro</i> qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and <i>in silico</i> analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (<i>MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2</i>, and <i>ELOVL1)</i> showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.
DOI: 10.6084/m9.figshare.11979693.v1
title: Chromatin structure regulates Cancer Specific Alternative Splicing Events in Primary HPV related Oropharyngeal squamous cell carcinoma

description: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumors. In this study, eleven ASEs (<i>GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331</i>, and <i>ELOVL1)</i> were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. <i>In vitro</i> qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and <i>in silico</i> analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (<i>MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2</i>, and <i>ELOVL1)</i> showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.
DOI: 10.6084/m9.figshare.12054021
title: Comparative analysis of genome-wide DNA methylation in <i>Neurospora</i>

description: DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes <i>Neurospora</i>. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related <i>Neurospora</i> species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (<i>N. crassa</i>) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of <i>Neurospora</i> are shaped by an evolutionary arms race between TEs and host defence.