Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq

Wedel, Carolin; Siegel, T. Nicolai

doi:10.1016/j.exppara.2017.03.003

Cited by 13 publications

(16 citation statements)

References 34 publications

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“…However, given that the responsible histone modifying enzymes are often unknown or tend to have multiple target sites, it has been difficult to unequivocally link individual histone modifications to specific biological functions. Recently we developed a quantitative proteomic-based approach to identify the target residues of histone acetyl transferases and deacetylases ( 42 ) and an MNase-ChIP-seq protocol for the detailed genome-wide mapping of individual histone modifications ( 24 ). In combination with these tools, the Cas9-based approach presented here represents an invaluable collection that should help unravel the role of histone modifications in trypanosomes.…”

Section: Discussionmentioning

confidence: 99%

“…Six microgram of DNA was suspended in 100 μl of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and fragmented using a Bioruptor Plus (Diagenode) at the following settings: low-power, 70 cycles of 30 s ‘on’ followed by 30 s ‘off’. Next, fragments between 100 and 300 bp were size-selected by gel electrophoresis and 40 ng were used to prepare indexed DNA libraries as described previously for DNA from ChIP assays ( 24 ). After purification, fragment sizes and library quality were analyzed in a Bioanalyzer 2100 (Agilent) and the DNA concentration was determined in a Qubit Fluorometer (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

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Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

Vasquez

Wedel

Cosentino

et al. 2018

Nucleic Acids Research

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Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR–Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR–Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3′UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

Vasquez

Wedel

Cosentino

et al. 2018

Nucleic Acids Research

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“…In brief, 2 × 10 8 cells were harvested, crosslinked in 1% formaldehyde, lysed using 200 µM digitonin (final concentration) and chromatin was fragmented using 1 U µl −1 MNase (Sigma-Aldrich), for details see ref. 65 . Immunoprecipitation was performed using Dynabeads M-280 sheep anti-rabbit IgG (Invitrogen) coupled to 10 µg polyclonal affinity-purified H2A.Z rabbit antibody 51 or using Dynabeads Protein G (Invitrogen) coupled to 10 µg monoclonal, purified BB2 mouse antibody 64 , overnight (~14 h) at 4°C in the presence of 0.05% SDS (final concentration).…”

Section: Methodsmentioning

confidence: 99%

Distinct roles for H4 and H2A.Z acetylation in RNA transcription in African trypanosomes

et al. 2020

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Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription. To disentangle these complex interdependent processes, we devised a highly sensitive strategy for quantifying histone acetylation levels at specific genomic loci. Taking advantage of the unusual genome organization in Trypanosoma brucei, we identified 58 histone modifications enriched at transcription start sites (TSSs). Furthermore, we found TSS-associated H4 and H2A.Z acetylation to be mediated by two different histone acetyltransferases, HAT2 and HAT1, respectively. Whereas depletion of HAT2 decreases H2A.Z deposition and shifts the site of transcription initiation, depletion of HAT1 does not affect H2A.Z deposition but reduces total mRNA levels by 50%. Thus, specifically reducing H4 or H2A.Z acetylation levels enabled us to reveal distinct roles for these modifications in H2A.Z deposition and RNA transcription.

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“…Immunoprecipitated DNA samples were sequenced in paired-end mode using an Illumina HiSeq 2500 or an Illumina NextSeq 500 sequencer with 2 × 100 and 2 × 76 cycles, respectively. The processing of the sequencing data was performed as described previously (Wedel & Siegel, 2017). In brief, the trimmed and clipped reads were mapped with bowtie2 version 2.1.0 (Langmead & Salzberg, 2012) or bowtie version 1.1.1 (Langmead et al, 2009) to the reference genomes Tb927v24 or Tb427v24 with different settings listed in Dataset EV2.…”

Section: Mapping Normalization and Visualization Of Sequencing Datamentioning

confidence: 99%

GT ‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

Förstner²,

et al. 2017

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Genome‐wide transcription studies are revealing an increasing number of “dispersed promoters” that, unlike “focused promoters”, lack well‐conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well‐defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site‐specific and genome‐wide assays, we identified GT‐rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context‐dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.

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Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq

Cited by 13 publications

References 34 publications

Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

Distinct roles for H4 and H2A.Z acetylation in RNA transcription in African trypanosomes

GT ‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

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