Harnessing enhanced nucleotide chemistry and toehold nanotechnology to reveal lncRNA spreading on chromatin
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ABSTRACT:
PROVIDER: PRJNA492138 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Understanding the targeting and spreading patterns of lncRNAs on chromatin requires a technique that can detect both high intensity binding sites and reveal genome-wide spreading patterns with high confidence. We developed an improved hybridization capture protocol to determine lncRNA localization using biotinylated LNA-containing oligonucleotides that hybridize to the target RNA and enhance capture specificity by including a protecting oligonucleotide that competitively displaces contaminating species, leading to highly specific RNA capture. This approach revealed the spreading pattern of roX2, a lncRNA involved in dosage compensation in D. melanogaster, how this pattern relates to chromatin features, and how spreading of roX2 changes upon cellular stress. Upon heat shock, roX2 displays reduced spreading on X chromosome and surprising relocalization to sites on autosomes, revealing how this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of lncRNAs on chromatin. This SuperSeries is composed of the SubSeries listed below.
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Project description:Domains of heterochromatin play important roles in the maintenance and regulation of eukaryotic genomes. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates the existence of robust mechanisms that limit heterochromatin spreading and thereby avoid silencing of expressed genes. A number of specific sequence elements have been found to serve as barriers to heterochromatin spreading; however, the mechanisms by which spreading is curtailed are generally not well understood. Here we uncover a role for PAF complex component Leo1 in regulating heterochromatin cis-spreading. A genetic screen revealed that loss of Leo1 results in spreading of heterochromatin across a centromeric (IRC) boundary element in fission yeast. Similar heterochromatin spreading was seen upon deletion of other components of the PAF complex, but not other factors involved in transcription-coupled chromatin modification, indicating a specific role for the PAF complex in heterochromatin regulation. Loss of Leo1 is associated with reduced levels of H4K16 acetylation at the boundary, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1? cells, suggesting that Leo1 antagonises heterochromatin spreading by facilitating H4K16 acetylation. Interestingly, Leo1 also regulates heterochromatin spreading independently of boundaries, and loss of Leo1 causes redistribution of heterochromatin, in particular resulting in substantial expansion of telomeric heterochromatin domains. The PAF complex is known to be an important regulator of transcription-related chromatin modifications; our findings reveal a previously undescribed role for this complex in global regulation of heterochromatin spreading in cis. 8 samples: input (whole cell extract) and IP from H3K9me2 ChIP in wild-type and leo1? cells, in duplicate
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Project description:Domains of heterochromatin play important roles in the maintenance and regulation of eukaryotic genomes. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates the existence of robust mechanisms that limit heterochromatin spreading and thereby avoid silencing of expressed genes. A number of specific sequence elements have been found to serve as barriers to heterochromatin spreading; however, the mechanisms by which spreading is curtailed are generally not well understood. Here we uncover a role for PAF complex component Leo1 in regulating heterochromatin cis-spreading. A genetic screen revealed that loss of Leo1 results in spreading of heterochromatin across a centromeric (IRC) boundary element in fission yeast. Similar heterochromatin spreading was seen upon deletion of other components of the PAF complex, but not other factors involved in transcription-coupled chromatin modification, indicating a specific role for the PAF complex in heterochromatin regulation. Loss of Leo1 is associated with reduced levels of H4K16 acetylation at the boundary, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by facilitating H4K16 acetylation. Interestingly, Leo1 also regulates heterochromatin spreading independently of boundaries, and loss of Leo1 causes redistribution of heterochromatin, in particular resulting in substantial expansion of telomeric heterochromatin domains. The PAF complex is known to be an important regulator of transcription-related chromatin modifications; our findings reveal a previously undescribed role for this complex in global regulation of heterochromatin spreading in cis.
2015-05-22 | GSE61688 | GEO
Project description:We use small RNA sequencing to look for spreading of secondary and tertiary siRNAs along transcripts targeted by ectopic hairpin-derived primary siRNAs. We find that signals within the 3' UTR inhibit siRNA spreading. sequencing of 20-30nt sRNAs from Argonaute immunoprecipitation
2013-12-26 | E-GEOD-52534 | biostudies-arrayexpress
Project description:We use small RNA sequencing to look for spreading of secondary and tertiary siRNAs along transcripts targeted by ectopic hairpin-derived primary siRNAs. We find that signals within the 3' UTR inhibit siRNA spreading.
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Project description:Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain and constrained its aberrant spreading. Interestingly, aberrant spreading of the 5-kb heterochromatin domain observed in the dmm-1KO strain is accompanied by the sharp deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5-kb heterochromatin domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5-kb heterochromatin domain. More importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin. Altogether, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.
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