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Series GSE59980 Query DataSets for GSE59980
Status Public on Aug 01, 2014
Title Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Methylation profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Summary Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consist- ing of multiple zinc finger domains are currently be- ing developed for clinical applications. However, no genome-wide investigations into their binding speci- ficity have been performed. We have created six- finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB do- main (SKD) that interacts with a complex contain- ing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5- fold, with a majority of the new sites located out- side of promoters. De novo motif analyses suggest that the lack of binding specificity is due to sub- sets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger bind- ing sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.
 
Overall design ATF plasmids were designed and stably integrated into MCF7 cells as described in (Stolzenburg et al. 2012). Stable lines were grown at 30–80% confluency in Dulbecco’s Modified Eagle’s Medium (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin; cells were selected using 5 µg/ml puromycin (VWR, Radnor, PA) and 200 µg/ml G418 (VWR, Radnor, PA). ATF expression was induced by treatment with media containing 1µg/ml doxycycline (VWR, Radnor, PA) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h. ATF expression was confirmed by hemagglutinin (HA) tag western blot prior to HA ChIP-seq, histone ChIP-seq and RNA-seq analysis. For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 mL low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 × 10∧7 cells/mL. Cells are then sonicated to a fragment size range of 500–800 bp. Samples were then diluted in 1 mL low-salt buffer and incubated with 3 µL of anti-HA antibody (Covance, Princeton, NJ). Three-hundred microliter Sepharose A beads (GE Healthcare Life Science) were used for pull-down. Samples were sequenced at the UNC-CH Genome Analysis Facil- ity (Chapel Hill, NC) on an HiSeq (Illumina, San Diego, CA) to read counts of 4.1–67.3 M total reads. For histone ChIP-seq, antibodies to H3K4me3 (Cell Signaling Tech- nologies CST9751), H3K9Ac (Cell Signaling Technologies CST9649) and H3K9me3 (Diagenode pAb-056-050) were used; samples were prepared as previously described (O’Geen et al. 2011).
 
Contributor(s) Grimmer MR, Stolzenburg S, Ford E, Lister R, Blancafort P, Farnham PJ
Citation(s) 25122745
Submission date Jul 31, 2014
Last update date May 15, 2019
Contact name Matt Grimmer
E-mail(s) grimmer@usc.edu
Organization name USC
Department Biochemistry
Lab Peggy Farnham
Street address 1450 Biggy Street NRT6514
City Los Angeles
State/province CA
ZIP/Postal code 90033
Country USA
 
Platforms (1)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
Samples (25)
GSM1462953 HA_ChIP_Empty_vector_rep1
GSM1462954 HA_ChIP_Empty_vector_rep2
GSM1462955 HA_ChIP_552_rep1
Relations
BioProject PRJNA257209
SRA SRP045155

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
GSE59980_552-SKD_HC.gff.gz 305.3 Kb (ftp)(http) GFF
GSE59980_552_HC.gff.gz 48.7 Kb (ftp)(http) GFF
GSE59980_598-SKD_HC.gff.gz 309.3 Kb (ftp)(http) GFF
GSE59980_598-SKD_K4me3+Dox.gff.gz 461.2 Kb (ftp)(http) GFF
GSE59980_598-SKD_K4me3-Dox.gff.gz 506.4 Kb (ftp)(http) GFF
GSE59980_598-SKD_K9ac+Dox.gff.gz 117.8 Kb (ftp)(http) GFF
GSE59980_598-SKD_K9ac-Dox.gff.gz 39.1 Kb (ftp)(http) GFF
GSE59980_598-SKD_K9me3+Dox.gff.gz 94.2 Kb (ftp)(http) GFF
GSE59980_598-SKD_K9me3-Dox.gff.gz 192.0 Kb (ftp)(http) GFF
GSE59980_598_HC.gff.gz 73.9 Kb (ftp)(http) GFF
GSE59980_RNA-seq_598-SKD.txt.gz 5.1 Mb (ftp)(http) TXT
GSE59980_WGBS_598-SKD.bed.gz 184.7 Mb (ftp)(http) BED
GSE59980_pT3_peaks.gff.gz 16.5 Kb (ftp)(http) GFF
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