Journal: Epigenetics & Chromatin

Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

doi: 10.1186/s13072-018-0201-5

Figure Lengend Snippet: Characterization of 27nt-RNAs and their association with macronuclear sequences. a Time course of small RNA fragment size distribution (10–50nt) and associated stages of macronuclear development showing the dominance of 27nt—RNAs (light blue columns). Red columns mark 21–22nt-RNAs. The y-axis shows the normalized read quantity (× 10 5 ). b The illustration shows the coverage of 27nt-RNAs (light blue bars), 21–22nt-RNAs (red bars) and mRNA reads (green curves) on four exemplary nanochromosomes with complete telomere-to-telomere sequence (red contigs). Notably, the mapped area frequently includes introns (1.95% of 27nt-RNA/1.43% of 21–22nt-RNA). The position of predicted full-length transcripts (yellow arrows) and coding sequences (CDS; green arrows, occasionally interrupted by introns [red arrows]) is illustrated. The bars below (purple) show furthermore the coverage of 27nt-RNAs, which were purified from pulled-down RNA/DNA hybrids using the mouse anti-RNA/DNA hybrid [S9.6] monoclonal antibody (mAb). c Average sncRNA (27nt-RNAs and 21–22nt-RNAs) read counts matching per contig. d Combined correlation analyses between mRNA reads (transcript), 27nt-RNAs and the relative nanochromosome copy numbers. e PAR-CLIP revealed a prominent RNA band well in agreement with the expected size of a PIWI1/27nt-RNA complex (sncRNA1)), whereas a smaller, at best very faint band could correspond to the expected size of a PIWI1/21–22nt-RNA complex (sncRNA2). f The read coverage is shown on the left for 4 exemplary nanochromosomes for 27nt-RNAs enriched in immunocomplexes using the mouse anti-RNA/DNA hybrid [S9.6] mAb (Kerafast #ENH001) (purple signals) or, respectively, rabbit anti-PIWIL1 polyclonal antibody (pAb) (Abcam #ab12337) (light blue signals). Symbolism and colour coding of the nanochromosome annotation equals ( b ). The chart (right) shows the results of correlation analyses between the reads obtained from 27nt-RNA-seq ( x -axis: RNA/DNA-IP; y -axis: PIWI1-IP)

Article Snippet: For immunoprecipitation 3 µg nucleic acids per sample were incubated overnight with 2.5 µg anti-RNA/DNA hybrid [S9.6] mouse monoclonal antibody and 25 µL magnetic protein A beads (Diagenode).

Techniques: Sequencing, Purification, RNA Sequencing Assay

Journal: Epigenetics & Chromatin

Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

doi: 10.1186/s13072-018-0201-5

Figure Lengend Snippet: Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electrophoresis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana (reviewed in ). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted

Article Snippet: For immunoprecipitation 3 µg nucleic acids per sample were incubated overnight with 2.5 µg anti-RNA/DNA hybrid [S9.6] mouse monoclonal antibody and 25 µL magnetic protein A beads (Diagenode).

Techniques: Agarose Gel Electrophoresis, Amplification, Sequencing, Purification

Journal: Epigenetics & Chromatin

Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

doi: 10.1186/s13072-018-0201-5

Figure Lengend Snippet: The nanochromosome copy number in vegetative macronuclei is an indicator for the existence of ‘RNA-induced DNA replication interference’ following S-phase treatments. a Replication in vegetative macronuclei (M) takes place in replication bands comprising of a forward zone (fz) with high DNA concentration and a rear zone (rz) with low DNA concentrations. The fz determines the direction of migration (arrow). Replication bands do never appear in micronuclei (m). b The motility of chromatin in macronuclei is strictly constrained, as revealed through pulse-chase-pulse experiments, where newly replicated DNA was 30 min pulse-labelled with 5-iodo-2′-deoxyuridine (red signals), followed by a 2 h chase and then a 30 min pulse with 5-chloro-2′-deoxyuridine (yellow signals): The signals from the first pulse remain within a restricted area even after prolonged observation periods/chases . The arrow indicates the migration direction. a , b Top-Pro-3 was used for DNA counterstaining, false colours were used for illustration. c The chart illustrates the effects of mock/bait RNA treatment (microinjection) on the copy numbers of targeted nanochromosomes at 3 different time periods ( t 1–3 ). The chart combines the data from all (target) experiments. The mock median was used as reference (onefold change) for the calculations of relative fold changes. Data was obtained from 48 independent qPCR reactions in triplicate (mock RNA) or 64 independent qPCR reactions in triplicate (bait RNA), whereby three different reporter amplicons were evenly distributed for bait RNA PCR reactions

Article Snippet: For immunoprecipitation 3 µg nucleic acids per sample were incubated overnight with 2.5 µg anti-RNA/DNA hybrid [S9.6] mouse monoclonal antibody and 25 µL magnetic protein A beads (Diagenode).

Techniques: Concentration Assay, Migration, Pulse Chase

Journal: Epigenetics & Chromatin

Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

doi: 10.1186/s13072-018-0201-5

Figure Lengend Snippet: The electron micrograph (kindly donated by Ada and Don Olins) below shows a section from a polytene chromosome from developing macronuclei in Stylonychia . Tight heterochromatic bands are interconnected by uncondensed chromatin fibres. The right-handed cartoon is an interpretation of that micrograph within a model for ‘RNA-induced DNA replication interference’ through 27nt-RNAs. 27nt-RNAs protect DNA from being lost during macronuclear development. Accordingly, PIWI1/27nt-RNA complexes block polytenization of covered MDSs, thus limiting the replication-dependent de novo deposition of nucleosomes. Histone variant H3.4 is probably permissive for H3K27me3. H3K27me3 nucleosomes become enriched during a restricted timeframe. Eventually, this tight spatiotemporal coordination leads to the association of H3(.4)K27me3 preferentially with bulk DNA—besides H3.5 that is continuously expressed. The heterochromatin formation via H3K27me3 is limited to those regions not protected by PIWI1/27nt-RNA complexes

Article Snippet: For immunoprecipitation 3 µg nucleic acids per sample were incubated overnight with 2.5 µg anti-RNA/DNA hybrid [S9.6] mouse monoclonal antibody and 25 µL magnetic protein A beads (Diagenode).

Techniques: Blocking Assay, Variant Assay