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Series GSE62880 Query DataSets for GSE62880
Status Public on May 12, 2015
Title FACT and Spt6 Prevent Cryptic Transcription by Impeding H2A.Z Loading in Gene Bodies
Organism Saccharomyces cerevisiae
Experiment type Genome binding/occupancy profiling by genome tiling array
Summary H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription.
 
Overall design We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
 
Contributor(s) Jeronimo C, Watanabe S, Kaplan CD, Peterson CL, Robert F
Citation(s) 25959393
Submission date Oct 30, 2014
Last update date Aug 30, 2018
Contact name Francois Robert
E-mail(s) francois.robert@ircm.qc.ca
Organization name IRCM
Lab Chromatin and Genomic Expression
Street address 110 av des Pins Ouest
City Montreal
State/province QC
ZIP/Postal code H2W 1R7
Country Canada
 
Platforms (2)
GPL4131 Agilent-014810 Yeast Whole Genome ChIP-on-Chip Microarray 4x44K (G4493A)
GPL18340 Agilent-034462 Yeast_4x180K
Samples (116)
GSM1535478 abHtz1vsabH2B_ino80DyFR933_rep1
GSM1535479 abHtz1vsabH2B_ino80DyFR933_rep2
GSM1535480 abHtz1vsabH2B_rsc14DyFR940_rep1
Relations
BioProject PRJNA265882

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE62880_RAW.tar 4.2 Gb (http)(custom) TAR (of GPR)
Processed data included within Sample table
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