Biology subjects

Johnston, A. D.

Publications and source records attributed to Johnston, A. D..

5 recordsLinked to original sources

bioRxiv2024

Unraveling cfDNA Fragmentation Patterns: Linking Chromatin Architecture to Cancer Diagnostic Sensitivity

Circulating cell-free DNA (cfDNA) liquid biopsies offer a powerful, non-invasive approach for cancer diagnostics, leveraging DNA fragments primarily released during apoptosis. Using droplet digital PCR on cfDNA samples from 88 breast and colorectal cancer patients, we uncovered highly consistent nucleosome breakpoints across individuals. Proximity to these cfDNA breakpoints diminishes the sensitivity of somatic mutation assays, including those targeting IDH1 and NRAS hotspot mutations, underscoring the importance of precise nucleosome positioning data. We introduce a probabilistic nucleosome protection scoring method that enables high-resolution mapping of nucleosome positions and cfDNA breakpoints. These maps reveal distinct nucleosome spacing patterns between heterochromatin and euchromatin, corresponding to different chromatin fiber topologies. Analysis of X-chromosome inactivation further supports the topoisomer model: heterochromatins 187 bp nucleosome repeat length differs from euchromatins 182 bp, generating wave interference patterns in nucleosome signals. Our findings suggest that loop-mediated transitions from T2 to T1 topoisomers shape chromatin accessibility and cfDNA fragmentation, advancing the diagnostic potential of liquid biopsy assays. HighlightsO_LINucleosome breakpoint conservation impacts somatic mutation assay sensitivity C_LIO_LIProbabilistic protection scoring enhances cfDNA nucleosome peak resolution C_LIO_LIChromatin states exhibit unique nucleosome spacing linked to distinct topologies, influencing cfDNA fragmentation C_LIO_LIChromosome X-inactivation phasing analyses reveal waveform interference in nucleosome positioning signals C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/623159v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@65893corg.highwire.dtl.DTLVardef@14c986org.highwire.dtl.DTLVardef@ba9306org.highwire.dtl.DTLVardef@10a7364_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2019

A cellular stress response induced by the CRISPR/dCas9 activation system is not heritable through cell divisions

The CRISPR/Cas9 system can be modified to perform epigenetic editing by utilizing the catalytically-inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions - an epigenetic regulatory property. Here we show how distinct components of this CRISPR/dCas9 activation system, particularly untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR/dCas9 components in human cells, which is transient and not maintained through cell divisions.

genetics↗

bioRxiv2018

High efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells.

While lymphoblastoid cell lines (LCLs) represent a valuable resource for population genetic studies, they are usually regarded as difficult for CRISPR-mediated genomic editing. It would be valuable to be able to take the results of their functional variant studies and test them in the same LCLs. We describe a protocol using a single-stranded donor oligonucleotide (ssODN) strategy for scarless editing in LCLs. The protocol involves optimized transfection, flow cytometric sorting of transfected cells to single cells in multi-well plates and growth in conditioned, serum-rich medium, followed by characterization of the clones. Amplicon sequencing reveals the relative proportions of alleles with different editing events, with sequencing of DNA from clones showing the frequencies of events in individual cells. We find 12/60 (20%) of clones selected in this manner to have the desired ssODN-mediated recombination event. Long-range PCR of DNA at the edited locus and of RT-PCR products for the gene traversing the edited locus reveals 3/6 characterized clones (50%) to have large structural mutations of the region that are missed by sequencing just the edited site. The protocol does not require the use of lentiviruses or stable transfection, and makes LCLs a realistic cell type for consideration for CRISPR-mediated genomic targeting.

genomics↗

bioRxiv2017

WHOLE GENOME BISULPHITE SEQUENCING USING THE ILLUMINA HISEQ X SYSTEM

The Illumina HiSeq X platform has helped to reduce the cost of whole genome sequencing substantially, but its application for bisulphite sequencing is not straightforward. We describe the optimization of a library preparation and sequencing approach that maximizes the yield and quality of sequencing, and the elimination of a previously unrecognized artefact affecting several percent of bisulphite sequencing reads.

genomics↗

bioRxiv2017

Intragenomic Redistribution Of Host Transcription Factor Binding With Toxoplasma gondii Infection

The intracellular pathogen Toxoplasma gondii modifies a number of host cell processes. The mechanisms by which T. gondii alters host gene expression are incompletely understood. This study focuses on how the regulators of gene expression in human host cells respond to T. gondii 24 hours following infection to cause specific patterns of transcriptional dysregulation. The most striking finding was the altered landscape of transposase-accessible chromatin by infection. We found both gains and losses of loci of open chromatin enriched in proximity to transcriptionally altered genes. Both DNA sequence motif analysis at the loci changing chromatin accessibility and network analysis of the genes with transcription and regulatory changes implicate a central role for the AP-1 transcription factor. We validated the redistribution of AP-1 in the host genome using chromatin immunoprecipitation studies of the c-Fos component of AP-1. As infection with T. gondii is associated with the cell failing to progress through the cell cycle, all of the changes observed occur in the absence of cell division and within 24 hours, an insight into the dynamism of these transcriptional regulatory events. We conclude that T. gondii infection influences transcriptional regulation through transcription factor re-targeting to modify the cis-regulatory landscape of the host nucleus.\n\nAUTHOR SUMMARYThe complex interactions between the intracellular pathogen Toxoplasma gondii and the host cell manifest as expression changes of host genes. T. gondiis secreted effectors have been extensively studied and include factors that influence the properties of transcription factors, resulting in post-translational modifications and changes in intracellular localization. To gain insights into how T. gondii exerts specific influences on host transcriptional regulation, we used genome-wide approaches to study gene expression, cytosine modifications, and chromatin structure of the host cell 24 hours after infection. The greatest insights were gained from the mapping of loci of transposase-accessible chromatin, revealing a consistently altered pattern of a subset of loci becoming inaccessible, with the simultaneous acquisition of a new set of infection-associated loci of open chromatin. The sequences at these loci were enriched for certain transcription factor binding motifs, in particular that of AP-1, the transcription factor formed by c-Jun and c-Fos heterodimers. Network analysis revealed a central role for c-Jun and c-Fos in the infection-associated perturbations, prompting a chromatin immunoprecipitation approach that confirmed the redistribution of c-Fos in infected cells. We conclude that a T. gondii infection leads to an intragenomic redistribution of host transcription factor binding, with resulting effects on host gene expression.

genomics↗