Dataset Information

The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila.

ABSTRACT: Advances in genomic technology led to a more focused pattern for the distribution of chromosomal proteins and a better understanding of their functions. The recent development of the CUT&RUN technique marks one of the important such advances. Here we develop a modified CUT&RUN technique that we termed nanoCUT&RUN, in which a high affinity nanobody to GFP is used to bring micrococcal nuclease to the binding sites of GFP-tagged chromatin proteins. Subsequent activation of the nuclease cleaves the chromatin, and sequencing of released DNA identifies binding sites. We show that nanoCUT&RUN efficiently produces high quality data for the TRL transcription factor in Drosophila embryos, and distinguishes binding sites specific between two TRL isoforms. We further show that nanoCUT&RUN dissects the distributions of the HipHop and HOAP telomere capping proteins, and uncovers unexpected binding of telomeric proteins at centromeres. nanoCUT&RUN can be readily applied to any system in which a chromatin protein of interest, or its isoforms, carries the GFP tag.

SUBMITTER: Chen T

PROVIDER: S-EPMC9473618 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila.

Chen Tao T Wei Xiaolu X Courret Cécile C Cui Min M Cheng Lin L Wu Jing J Ahmad Kami K Larracuente Amanda M AM Rong Yikang S YS

PLoS genetics 20220901 9

Advances in genomic technology led to a more focused pattern for the distribution of chromosomal proteins and a better understanding of their functions. The recent development of the CUT&RUN technique marks one of the important such advances. Here we develop a modified CUT&RUN technique that we termed nanoCUT&RUN, in which a high affinity nanobody to GFP is used to bring micrococcal nuclease to the binding sites of GFP-tagged chromatin proteins. Subsequent activation of the nuclease cleaves the ...[more]

PMID: 36048878

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Project description:BackgroundTelomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline.ResultsTo fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins.ConclusionspiRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline.

Project description:Cleavage under targets and release using nuclease (CUT&RUN) is a recently developed chromatin profiling technique that uses a targeted micrococcal nuclease cleavage strategy to obtain high-resolution binding profiles of protein factors or to map histones with specific post-translational modifications. Due to its high sensitivity, CUT&RUN allows quality binding profiles to be obtained with only a fraction of the starting material and sequencing depth typically required for other chromatin profiling techniques such as chromatin immunoprecipitation. Although CUT&RUN has been widely adopted in multiple model systems, it has rarely been utilized in Caenorhabditis elegans, a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging in C. elegans due to the toughness of the worm's cuticle and the sensitivity of the cells themselves. Here, we describe a robust CUT&RUN protocol for use in C. elegans to determine the genome-wide localization of protein factors and specific histone marks. With a simple protocol utilizing live, uncrosslinked tissue as the starting material, performing CUT&RUN in worms has the potential to produce physiologically relevant data at a higher resolution than chromatin immunoprecipitation. This protocol involves a simple dissociation step to uniformly permeabilize worms while avoiding sample loss or cell damage, resulting in high-quality CUT&RUN profiles with as few as 100 worms and detectable signal with as few as 10 worms. This represents a significant advancement over chromatin immunoprecipitation, which typically uses thousands or hundreds of thousands of worms for a single experiment. The protocols presented here provide a detailed description of worm growth, sample preparation, CUT&RUN workflow, library preparation for high-throughput sequencing, and a basic overview of data analysis, making CUT&RUN simple and accessible for any worm lab. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth and synchronization of C. elegans Basic Protocol 2: Worm dissociation, sample preparation, and optimization Basic Protocol 3: CUT&RUN chromatin profiling Alternate Protocol: Improving CUT&RUN signal using a secondary antibody Basic Protocol 4: CUT&RUN library preparation for Illumina high-throughput sequencing Basic Protocol 5: Basic data analysis using Linux.

Dataset Information

The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila.

Publications

The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila.

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