scholarly journals Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

10.3791/57080 ◽  
2017 ◽  
Author(s):  
Meagan Jezek ◽  
Alison Jacques ◽  
Deepika Jaiswal ◽  
Erin M. Green
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation

scholarly journals S-phase-independent silencing establishment in Saccharomyces cerevisiae

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Davis Goodnight ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Histone Modifications ◽  
Single Molecule ◽  
Chromatin Immunoprecipitation ◽  
S Phase ◽  
Cell Cycles ◽  
Identical Cell ◽  
Protein Recruitment ◽  
Molecular Nature

The establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using high-resolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that the cell-cycle-regulated step was downstream of Sir protein recruitment. In contrast to prior results, HML and HMR had identical cell-cycle requirements for silencing establishment, with no apparent contribution from a tRNA gene adjacent to HMR. We identified the cause of the S-phase requirement for silencing establishment: removal of transcription-favoring histone modifications deposited by Dot1, Sas2, and Rtt109. These results revealed that silencing establishment was absolutely dependent on the cell-cycle-regulated interplay between euchromatic and heterochromatic histone modifications.

scholarly journals S-phase independent silencing establishment in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Davis Goodnight ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Histone Modifications ◽  
Single Molecule ◽  
Chromatin Immunoprecipitation ◽  
S Phase ◽  
Cell Cycles ◽  
Identical Cell ◽  
Protein Recruitment ◽  
Molecular Nature

ABSTRACTThe establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using high-resolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that the cell-cycle-regulated step was downstream of Sir protein recruitment. In contrast to prior results, HML and HMR had identical cell-cycle requirements for silencing establishment, with no apparent contribution from a tRNA gene adjacent to HMR. We identified the cause of the S-phase requirement for silencing establishment: removal of transcription-favoring histone modifications deposited by Dot1, Sas2, and Rtt109. These results revealed that silencing establishment was absolutely dependent on the cell-cycle-regulated interplay between euchromatic and heterochromatic histone modifications.

scholarly journals Binding of an X-Specific Condensin Correlates with a Reduction in Active Histone Modifications at Gene Regulatory Elements

Genetics ◽  
2019 ◽  
Vol 212 (3) ◽  
pp. 729-742 ◽  
Author(s):  
Lena Annika Street ◽  
Ana Karina Morao ◽  
Lara Heermans Winterkorn ◽  
Chen-Yu Jiao ◽  
Sarah Elizabeth Albritton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Protein Complexes ◽  
Regulatory Elements ◽  
Evolutionarily Conserved ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Gene Regulatory Elements ◽  
Gene Regulatory ◽  
Regulatory Sites

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In Caenorhabditis elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing. Across the X, the DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. The DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.

scholarly journals Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

2021 ◽  
Vol 12 ◽  
Author(s):  
Weizhi Ouyang ◽  
Xiwen Zhang ◽  
Yong Peng ◽  
Qing Zhang ◽  
Zhilin Cao ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Protein A ◽  
Transcriptional Factor ◽  
Experimental Procedure ◽  
Histone Marks ◽  
Genome Wide ◽  
Efficient Alternative ◽  
Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

scholarly journals The Inheritance of Histone Modifications Depends upon the Location in the Chromosome in Saccharomyces cerevisiae

PLoS ONE ◽  
2011 ◽  
Vol 6 (12) ◽  
pp. e28980 ◽  
Author(s):  
Hiroshi Masumoto ◽  
Ryuichiro Nakato ◽  
Masato Kanemaki ◽  
Katsuhiko Shirahige ◽  
Mayumi Hachinohe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Modifications

scholarly journals A Silencer Promotes the Assembly of Silenced Chromatin Independently of Recruitment

2008 ◽  
Vol 29 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Patrick J. Lynch ◽  
Laura N. Rusche
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Chromatin Immunoprecipitation ◽  
Protein Assembly ◽  
The Other ◽  
Sir Proteins ◽  
Genomic Locations ◽  
Nonlinear Fashion ◽  
Silent Mating Type Loci

ABSTRACT In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.

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