Biology subjects

Coruh, C.

Publications and source records attributed to Coruh, C..

8 recordsLinked to original sources

bioRxiv2023

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing.

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

molecular biology↗

bioRxiv2023

Breakage fusion bridge cycles drive high oncogene copy number, but not intratumoral genetic heterogeneity or rapid cancer genome change

Oncogene amplification is a major driver of cancer pathogenesis. Breakage fusion bridge (BFB) cycles, like extrachromosomal DNA (ecDNA), can lead to high copy numbers of oncogenes, but their impact on intratumoral heterogeneity, treatment response, and patient survival are not well understood due to difficulty in detecting them by DNA sequencing. We describe a novel algorithm that detects and reconstructs BFB amplifications using optical genome maps (OGMs), called OM2BFB. OM2BFB showed high precision (>93%) and recall (92%) in detecting BFB amplifications in cancer cell lines, PDX models and primary tumors. OM-based comparisons demonstrated that short-read BFB detection using our AmpliconSuite (AS) toolkit also achieved high precision, albeit with reduced sensitivity. We detected 371 BFB events using whole genome sequences from 2,557 primary tumors and cancer lines. BFB amplifications were preferentially found in cervical, head and neck, lung, and esophageal cancers, but rarely in brain cancers. BFB amplified genes show lower variance of gene expression, with fewer options for regulatory rewiring relative to ecDNA amplified genes. BFB positive (BFB (+)) tumors showed reduced heterogeneity of amplicon structures, and delayed onset of resistance, relative to ecDNA(+) tumors. EcDNA and BFB amplifications represent contrasting mechanisms to increase the copy numbers of oncogene with markedly different characteristics that suggest different routes for intervention.

cancer biology↗

bioRxiv2021

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we present a method for targeted purification of megabase-sized ecDNA by combining in-vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA (CRISPR-CATCH). We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells. Targeted purification of ecDNA versus chromosomal DNA enabled phasing of genetic variants and provided definitive proof of an EGFRvIII mutation on ecDNA and wild-type EGFR on chromosomal DNA in a glioblastoma neurosphere model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNA compared to the native chromosomal locus in the same cells. Finally, separation of ecDNA species by size and sequencing allowed accurate reconstruction of megabase- sized ecDNA structures with base-pair resolution. CRISPR-CATCH is a new addition to the toolkit for studying focal amplifications in cancer and will accelerate studies aiming to explore the genetic and epigenetic landscapes of ecDNA.

cancer biology↗

bioRxiv2021

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

genomics↗

bioRxiv2021

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation that are differentially expressed during plant development, play major roles in controlling tissue-specific DNA methylation patterns. Depending on the tissue, the genetic requirements for specific CLSYs differ significantly and, on a global scale, certain clsy mutants are sufficient to largely shift the epigenetic landscape between tissues. Together, these findings not only reveal substantial epigenetic diversity between tissues, but assign these changes to specific CLSY proteins, revealing how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

plant biology↗

bioRxiv2020

AmpliconReconstructor: Integrated analysis of NGS and optical mapping resolves the complex structures of focal amplifications in cancer

Oncogene amplification, a major driver of cancer pathogenicity, is often mediated through focal amplification of genomic segments. Recent results implicate extrachromosomal DNA (ecDNA) as the primary mechanism driving focal copy number amplification (fCNA) - enabling gene amplification, rapid tumor evolution, and the rewiring of regulatory circuitry. Resolving an fCNAs structure is a first step in deciphering the mechanisms of its genesis and the subsequent biological consequences. Here, we introduce a powerful new computational method, AmpliconReconstructor (AR), for integrating optical mapping (OM) of long DNA fragments (>150kb) with next-generation sequencing (NGS) to resolve fCNAs at single-nucleotide resolution. AR uses an NGS-derived breakpoint graph alongside OM scaffolds to produce high-fidelity reconstructions. After validating performance by extensive simulations, we used AR to reconstruct fCNAs in seven cancer cell lines to reveal the complex architecture of ecDNA, breakage-fusion-bridge cycles, and other complex rearrangements. By distinguishing between chromosomal and extrachromosomal origins, and by reconstructing the rearrangement signatures associated with a given fCNAs generative mechanism, AR enables a more thorough understanding of the origins of fCNAs, and their functional consequences.

bioinformatics↗

bioRxiv2019

Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants

Plant endogenous small RNAs (sRNAs) are important regulators of gene expression. There are two broad categories of plant sRNAs: microRNAs (miRNAs) and endogenous short interfering RNAs (siRNAs). MicroRNA loci are relatively well-annotated but comprise only a small minority of the total sRNA pool; siRNA locus annotations have lagged far behind. Here, we used a large dataset of published and newly generated sRNA sequencing data (1,333 sRNA-seq libraries containing over 20 billion reads) and a uniform bioinformatic pipeline to produce comprehensive sRNA locus annotations of 47 diverse plants, yielding over 2.7 million sRNA loci. The two most numerous classes of siRNA loci produced mainly 24 nucleotide and 21 nucleotide siRNAs, respectively. 24 nucleotide-dominated siRNA loci usually occurred in intergenic regions, especially at the 5-flanking regions of protein-coding genes. In contrast, 21 nucleotide-dominated siRNA loci were most often derived from double-stranded RNA precursors copied from spliced mRNAs. Genic 21 nucleotide-dominated loci were especially common from disease resistance genes, including from a large number of monocots. Individual siRNA sequences of all types showed very little conservation across species, while mature miRNAs were more likely to be conserved. We developed a web server where our data and several search and analysis tools are freely accessible at http://plantsmallrnagenes.science.psu.edu.

plant biology↗

bioRxiv2017

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs

First paragraphDodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite1, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production2. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation levels. Hosts with mutations in two of the targets supported significantly higher growth of C. campestris. Homologs of target mRNAs from diverse plants also have predicted target sites to induced C. campestris miRNAs, and the same miRNAs are expressed and active against host targets when C. campestris parasitizes a different host, Nicotiana benthamiana. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.

plant biology↗