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Series GSE80425 Query DataSets for GSE80425
Status Public on Apr 20, 2016
Title Genome-wide identification of H2A.Z-interacting proteins by bPPI-seq
Organism Mus musculus
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by high throughput sequencing
Other
Summary Histone variant H2A.Z is a critical player in setting up the chromatin environment that mediates transcription and other activities on chromatin. However, how H2A.Z is incorporated to specific chromatin regions is not clear. To examine the potential role of sequence-specific transcription factors in targeting H2A.Z, we screened for genome-wide H2A.Z-interacting proteins in vivo using a novel technique called bait Protein-Protein Interaction-sequencing (bPPI-seq). Among the hundreds of H2A.Z-interacting proteins identified by bPPI-seq, we show that a zinc-finger transcription factor, Osr1 interacts with H2A.Z both in vitro and in vivo and co-localizes with H2A.Z on chromatin. Knockdown of Osr1 compromised H2A.Z deposition to hundreds of chromatin sites enriched with Osr1 binding motifs. Furthermore, Osr1 and H2A.Z co-regulate the expression of numerous target genes. These results indicate that Osr1 directly interacts with H2A.Z, mediates its incorporation to a large number of target sites and regulates gene expression. Our data indicate that bPPI-seq can be widely applied to identify unbiasedly interacting proteins under physiologic conditions.
 
Overall design Examination of the H2A.Z-interacting proteins by a novel bait Protein-Protein Interaction-sequencing method

GSM2628249-GSM2628254: ReMTH-VNC 1,2 and 3 (3 different reading frames) and pEco were co-transfected into GP2 293 cells using Lipofectamine 2000 (Invitrogen). Supernatants containing packaged viral particles were collected 48h after transfection. 30 ml viral particles were used to infect 50 million 3T3 Tet-on 3G stable cell line expressing GFPN-H2A.Z, GFPN-H2A or GFPN alone to avoid multiple integrations. After 48 hours of infection, the cells were selected in DMEM containing 2μg/ml puromycin (Sigma) for 2 days and maintained in 1μg/ml puromycin for 3 days. Then equal number of cells infected with viruses for the 3 reading frames were pooled and induced by 2μg/ml Doxycycline for 48 hrs before sorting for green cells. GFP-positive cells were sorted with FACSAria (BD Biosciences).
 
Contributor(s) Zhang Y, Ku WL, Liu S, Cui K, Jin W, Tang Q, Lv W, Ni B, Zhao K
Citation(s) 28862252
Submission date Apr 19, 2016
Last update date Jul 25, 2021
Contact name Keji Zhao
E-mail(s) zhaok@nhlbi.nih.gov
Organization name national Institute of Health
Department National Heart, Lung, and Blood Institute
Street address Building 10 Room 7B06A
City Bethesda
State/province MD
ZIP/Postal code 20814
Country USA
 
Platforms (2)
GPL13112 Illumina HiSeq 2000 (Mus musculus)
GPL21493 Illumina HiSeq 3000 (Mus musculus)
Samples (28)
GSM2127092 ChIP-Seq mouse SELEX Osr1
GSM2127093 ChIP-seq mouse 3T3 shLuc H2A.Z(1)
GSM2127094 ChIP-seq mouse 3T3 shLuc H2A.Z(2)
Relations
BioProject PRJNA318889
SRA SRP073488

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Supplementary file Size Download File type/resource
GSE80425_RAW.tar 100.9 Mb (http)(custom) TAR (of BED, TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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