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Series GSE93755 Query DataSets for GSE93755
Status Public on Jun 21, 2018
Title Constrained pioneering and partner factor redirection by PU.1 shape early T-cell gene regulation
Organism Mus musculus
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by high throughput sequencing
Other
Summary A major problem with linking transcription factor binding to function is that many factors bind to a large number of, at least in any given cell type, seemingly irrelevant regions. This makes it hard to filter out which binding sites are responsible for the regulation of a given gene. PU.1 (Spi1, Sfpi1) is an excellent example of a transcription factor that works both to mediate developmental choices and to serve the alternative developmental fates that emerge from these choices. Its role in T-cell development is confined to the early stages where high PU.1 expression persists through multiple cell divisions and is sharply downregulated over the DN2a-to-DN2b T-cell commitment stage. Even though it is known that PU.1 is necessary for the survival of the earliest T-cell progenitors, where it is needed for optimal proliferation, control of alternative lineage genes, and correct timing of the access to T-lineage genes, it has not been well-studied how PU.1 finds it targets and exerts its functions within this context. Here, we show in detail how PU.1 selects its binding sites and subsequently regulate gene expression. We show that PU.1 has two effective affinity thresholds for occupancy depending on current chromatin openness as measured by ATAC-sequencing, but that it is easily capable of binding sites in closed chromatin. Unexpectedly, although its binding to promoters is least constrained, promoters are the only major class of sites where it exerts a predominantly negative effect; otherwise it works locally as an activator, mainly mediated through binding to sites in closed or dynamically closing distal enhancer elements, where it can rapidly open chromatin and induce histone acetylation. However, its ability to open the chromatin depends not only on its own affinity but also on the presence of collaborating factors, because we show that PU.1 introduction into PU.1-negative cells triggers a massive reorganization of occupancy patterns of at least three other factors: Runx1, Satb1, and Gata3. Most strikingly, PU.1’s “theft” of Runx1 and Satb1 from many sites where they were binding in the absence of PU.1 enables PU.1 to exert a novel form of repression even on genes where it has no binding sites itself. We show here that PU.1 requires domains outside of its DNA binding domain to properly open chromatin, and this structural requirement is directly connected with its ability to bind to Runx1 and to Satb1, and moreover, Runx1, in particular, is an important collaborator to activate many of its target genes in the context of early T-cell development. Thus, PU.1 regulates gene expression via two distinct mechanisms. First, PU.1 steals Satb1 and Runx1 from many genomic sites, thereby repressing T cell gene expression indirectly. Second, PU.1, opens chromatin, recruits Satb1 and Runx1 to new sites, and directly activates its target gene expression. In summary, we here present a model where a transcription factor can work through redeployment of other factors and not only through sites that it binds itself.
 
Overall design ChIP-Seq: Primary cell experiments: Briefly, lin- FLPs were expanded for 7 days on OP9-DL1, CD25+ enriched and retrovirally infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were co-cultured on OP9-DL1 for ~40h and then sorted as LiveGFP+CD25+ or LiveGFP+CD44+CD25- on BD FACSAriaTM. Scid.adh.2c2: HA-tagged-ChIP: Scid.adh.2c2 cells were infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were expanded for ~40h and then sorted as LiveGFP+on BD FACSAriaTM. 4-OHT experiment: Scid.adh.2c2 cells were transduced with Lzr-PU.1ert2 (Scid.adh.2c2-PU.1ert2) or Lzr-ert2 (Scid.adh.2c2-ert2) control, cells were expanded for 48h and infected (GFP+) cells followed by sorting of GFP+ cells on BD FACSAriaTM and further expanded in vitro prior to 2, 8 or 24h activation with 0.1 μM 4-OHT (4-hydroxytamoxifen). Satb1, Runx1, Fog1 and GATA3 ChIP: Scid.adh.2c2 cells were infected with retroviral supernatant of pMxs-Myc-Flag-PU.1-IRES-hNGFR and cultured for 48h. Approximately 3-5 million primary sorted CD25+ -or 10 million Scid.adh.2c2 cells for ChIP were fixed at RT in 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min followed by additional 10 min after addition of formaldehyde (FA) up to 1% (Satb1, Runx1, Fog1 and Ets1) or 10 min of 1% FA only (PU.1, HA,-tag, GATA3, H3-modifications). The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed in ice-cold 1xHBSS (Gibco). Cell pellets were snap frozen on dry ice and stored in -80°C. For DSG+FA crosslinked cells, nuclei were isolated by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5% NP40) + protease inhibitor cocktail (PIC) (Roche) on ice. Pelleted nuclei were dissolved in 0.5% Lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8)) + PIC. FA crosslinked cells only were resuspended 1% Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8). Lysates were sonicated on a Bioruptor (Diagenode) for 18 cycles, max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to a new tube and chromatin was diluted 3X in 1xHBSS+PIC followed by 2xRIPA (2% Triton, 2mM EDTA, 200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% sodium deoxycholate, 0.1%SDS + PIC). Approximately 10 μg per 107 cells of antibody (Rabbit anti-PU.1 polyclonal IgG (sc-352x, Santa Cruz Biotechnology), Rabbit HA-probe polyclonal IgG (sc-805x, Santa Cruz Biotechnology), Rabbit anti-Ets1 polyclonal IgG (sc-350x, Santa Cruz Biotechnology), Mouse anti-Gata3 monoclonal IgG (a mixture of sc-268, Santa Cruz Biotechnology and MAB26051, R&D Systems), Rabbit anti-Satb1 monoclonal IgG (ab109122, Abcam), Rabbit anti-Runx1 polyclonal IgG (ab23980, Abcam), Goat anti-Fog1 polyclonal IgG (sc-9361, Santa Cruz Biotechnolog), Rabbit polyclonal anti-H3K27Ac (ab4729, Abcam), Rabbit polyclonal anti-H3K4me2 (07-030, Millipore) or Rabbit polyclonal anti-H3K27me3 (07-449, Millipore) were hybridized to Dynabeads M-280 Sheep anti- Rabbit or Dynabeads M-280 Sheep anti- Mouse or Dynabeads Protein A/G (Invitrogen), respectively in 1ml 1xRIPA+PIC for 4h, washed twice with 1xRIPA, resuspended in 100 μl 1xRIPA+PIC and added to the diluted chromatin. For polyclonal and monoclonal antibodies, 20 and 50 μl of Dynabeads were used per μg antibody, respectively. ChIP was performed over night at 4°C, and subsequently washed (1 time with 1 ml Low Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton-X, 2 mM EDTA, 50 mM Tris-Hcl pH8, 150 mM NaCl), 1 time with 1 ml High Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton-X, 2 mM EDTA, 50 mM Tris-Hcl pH8, 500 mM NaCl), 1 time with 1 ml LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Igepal-CA630, 1% natrium deoxycholate, 1 mM EDTA, 10 mM Tris-Hcl pH8), 2 times with 1 ml TE buffer (10 mM Tris–HCl, pH 8.0, 10 mM EDTA)) and eluted for 6 h to O/N at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (NEB #E6240) following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample.

RNA-seq: CD25+ cell experiments: Briefly, lin- FLPs were expanded for 7 days on OP9-DL1, CD25+ enriched and retrovirally infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were co-cultured on OP9-DL1 for ~40h and then sorted as LiveGFP+CD25+ or LiveGFP+CD44+CD25- on BD FACSAriaTM. Scid.adh.2c2: Scid.adh.2c2 cells were transduced with Lzr-PU.1ert2 (Scid.adh.2c2-PU.1ert2) or Lzr-ert2 (Scid.adh.2c2-ert2) control, cells were expanded for 48h and infected (GFP+) cells followed by sorting of GFP+ cells on BD FACSAriaTM and further expanded in vitro prior to 2, 8 or 24h activation with 0.1 μM 4-OHT (4-hydroxytamoxifen). Total RNA was isolated from 200.000-500.000 primary or Scid.adh.2c2 cells using RNAeasy MicroKit (Qiagen) according to manufacturer’s recommendations. Libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530) from ~1 μg of total RNA following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample.

ATAC-seq: Eighty thousand Scid.adh.2c2 or Scid.adh.2c2 transduced Lzr-PU.1-ert2 or Lzr-er2 control vector followed by 0.1 uM 4-hydroxytamoxifen stimulation, 18.4 thousand primary sorted thymic ETP or 50,000 thymic DN3 cells were washed in ice cold HBSS-HEPES prior to the assay. For the primary cells, the final amplified libraries were purified and size-selected (x1.2 bead-to-DNA ratio) using SPRIselect-beads (Beckman-Coultier). Libraries were single-end sequenced on a HiSeq2500 (Illumina) (primary cells) or a NextSeq500 (Scid.adh.2c2) and produced approximately 30-50 million reads per sample.

Please note that processed data was not provided for some ChIP-seq samples since the provided HOMER peak files are merged in various ways throughout the manuscript and then read counts from various combinations of the bed-files are used to generate the plots. Instead, information of the peak files (used to get the data) was provided in the corresponding sample description field. The listed chip-seq peak files (that have been used to drive reads) are not processed versions of those per se, but explain which files and how they were used in the data analysis using the HOMER software (http://homer.salk.edu/).
 
Contributor(s) Ungerbäck J, Hosokawa H, Wang X, Tanaka T, Strid T, Williams BA, Matsumoto M, Sigvardsson M, Nakayama KI, Rothenberg EV
Citation(s) 29924977
Submission date Jan 18, 2017
Last update date May 15, 2019
Contact name Ellen Rothenberg
E-mail(s) evroth@its.caltech.edu
Organization name California Institute of Technology
Department Division of Biology and Biological Engineering
Street address 1200 E. California Blvd
City Pasadena
State/province CA
ZIP/Postal code 91125
Country USA
 
Platforms (2)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
GPL19057 Illumina NextSeq 500 (Mus musculus)
Samples (188)
GSM2461489 17215.FLP_CD25+_EVGFP_aHA-ChIP-seq-rep1
GSM2461490 17632.FLP_CD25+_EVGFP_aHA-ChIP-seq-rep2
GSM2461491 17216.FLP_CD25+_EVGFP_aPU.1-ChIP-seq-rep1
Relations
BioProject PRJNA362299
SRA SRP096946

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
GSE93755_20159_20160_sgCtrl_PU1_IDR_peaks-top-set_15_mm9.annotated.txt.gz 2.0 Mb (ftp)(http) TXT
GSE93755_20186_20162_sgRunx1_PU1_IDR_peaks-top-set_15_mm9.annotated.txt.gz 1.9 Mb (ftp)(http) TXT
GSE93755_DN2_ATAC_sgCtrl_ATAC_peaks.txt.gz 3.6 Mb (ftp)(http) TXT
GSE93755_DN2_ATAC_sgCtrl_sgPU1.edgeR_diff_expression.txt.gz 14.3 Mb (ftp)(http) TXT
GSE93755_DN2_ATAC_sgPU1_ATAC_peaks.txt.gz 3.5 Mb (ftp)(http) TXT
GSE93755_FLPCD25_EVGFP_aPU1_HOMER_IDR_peaks.txt.gz 301.7 Kb (ftp)(http) TXT
GSE93755_FLPCD25_PU1ENGHA_aHA_HOMER_IDR_peaks.txt.gz 2.2 Mb (ftp)(http) TXT
GSE93755_FLPCD25_PU1ETSHA_aHA_HOMER_IDR_peaks.txt.gz 1.0 Mb (ftp)(http) TXT
GSE93755_FLPCD25_PU1WTHA_aHA_HOMER_IDR_peaks.txt.gz 3.9 Mb (ftp)(http) TXT
GSE93755_FLPCD25_PU1WTHA_aPU1_HOMER_IDR_peaks.txt.gz 2.8 Mb (ftp)(http) TXT
GSE93755_JU_HH_et_al_RNA-seq_raw_counts_table.txt.gz 2.6 Mb (ftp)(http) TXT
GSE93755_JU_HH_et_al_RNA-seq_rpkm_table.txt.gz 3.3 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_ATAC_peaks.txt.gz 3.2 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_EVert2_0h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 3.4 Kb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_EVert2_24h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 3.4 Kb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_Ets1_HOMER_IDR_peaks.txt.gz 515.0 Kb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_Mock_aFog1_HOMER_IDR_peaks.csv.gz 36.5 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_Mock_aGATA3_HOMER_IDR_peaks.csv.gz 663.4 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_Mock_aRunx1_HOMER_IDR_peaks.csv.gz 505.7 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_Mock_aSatb1_HOMER_IDR_peaks.csv.gz 378.4 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_PU.1_aFog1_HOMER_IDR_peaks.csv.gz 17.7 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_PU.1_aGATA3_HOMER_IDR_peaks.csv.gz 615.7 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_PU.1_aRunx1_HOMER_IDR_peaks.csv.gz 463.8 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_PU.1_aSatb1_HOMER_IDR_peaks.csv.gz 396.0 Kb (ftp)(http) CSV
GSE93755_Scid.adh.2c2_PU1ENGHA_aHA_HOMER_IDR_peaks.txt.gz 1.1 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1ETSHA_aHA_HOMER_IDR_peaks.txt.gz 1.2 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1WTHA_aHA_HOMER_IDR_peaks.txt.gz 3.6 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1WTHA_aPU1_HOMER_IDR_peaks.txt.gz 2.9 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1ert2_0h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 431.6 Kb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1ert2_24h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 1.7 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1ert2_2h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 2.0 Mb (ftp)(http) TXT
GSE93755_Scid.adh.2c2_PU1ert2_8h_4-OHT_aPU1_HOMER_IDR_peaks.txt.gz 2.3 Mb (ftp)(http) TXT
GSE93755_Thy_DN3.ATAC_peaks.txt.gz 2.2 Mb (ftp)(http) TXT
GSE93755_Thy_ETP.ATAC_peaks.txt.gz 1.9 Mb (ftp)(http) TXT
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