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| Status |
Public on Feb 07, 2022 |
| Title |
DNA sequence-dependent heterochromatin microdomain formation |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Cell-type specific gene expression programs are established by distinct chromatin state patterns that involve thousands of heterochromatin microdomains of ~1-2 kb in size marked by di- and trimethylation of histone H3 at lysine 9 (H3K9me2/me3). However, no theoretical framework exists to predict the location and boundaries of such domains from the DNA sequence. Here, we compare H3K9me2/me3-heterochromatin microdomains in mouse embryonic stem cells (ESCs) that are dependent on the histone methylases SUV39H1/2 and GLP, transcription factor ADNP or chromatin remodeler ATRX. By applying a novel Ising-type chromatin hierarchical lattice (ChromHL) model, we identify two different microdomain types that are distinct with respect to their dependence on DNA sequence motifs or nucleosome interactions. ChromHL is able to predict microdomain location, extension and boundaries based on binding sites of PAX3, PAX9, ADNP, CTCF and repeat sequence motifs, the concentration of heterochromatin protein 1 (HP1) and the strength of nucleosome-nucleosome interactions. Thus, our result provides insight how distinct patterns of silenced heterochromatin states are implemented and regulated.
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| Overall design |
Wild type murine embryonic stem cell (ESC) wt26 and two ATRX knock out cell lines (KO1-40 and KO1-45) were constructed as described previously (Sadic et al., EMBO Rep, 2015). Cells were cultured on 0.2% v/v gelatine (in PBS) in high glucose DMEM (gibco 31053-028) supplemented with 1 mM sodium pyruvate and 4 mM L-Glutamine (PAA M11-006), 15% v/v FCS (Sigma F7524, lot: 091M3398), 1% v/v Penicillin-Streptomycin (PAN Biotech P06-07100), 100 µM β-mercaptoethanol (Sigma 63689), 1% v/v non-essential amino acids and 0.41% v/v LIF (self-made; supernatant from LIF-producing cells; batch: 7/26/14). Chromatin immunoprecipitation. Cells were fixed in 1% formaldehyde (Thermo Scientific 28906) in PBS for 10 minutes and stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with PBS/0.5 mM PMSF, dissolved in swelling buffer (25 mM Hepes pH7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, protease inhibitor COMPLETE from Roche) and incubated on ice for 10 minutes. After pelleting they were digested with MNase at 37°C for 15 minutes in a buffer containing 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris/HCl pH7.4 and 1x protease inhibitor from Cell Signalling. Chromatin was fragmented with a Covaris S2 sonicator (parameters: 900 s, burst 200, cycle 20%, intensity 8) in sonication buffer (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate) and centrifuged at 13000 rpm and 4°C for 15 minutes. The supernatant was mixed with 4 µg normal rabbit IgG (R&D Systems, AB-105-C, lot: ER1212071) and incubated with ChIP-grade protein G magnetic beads (Cell Signalling 9006S, 25 µl/sample) at 4°C for 1:45 h (pre-clearance). 1/20 of the supernatant was used as input sample and the remaining material was used for the IP reaction by adding 4 µg of anti-H3K9me3 antibody (Abcam, ab8898, lot: GR148830-2). The sample was incubated for 2 h at 4°C, protein G magnetic beads were added and incubated at 4°C overnight. On the next day, the IP samples were washed with sonication buffer, high-salt buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), Li buffer (20 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and finally twice with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Subsequently, the DNA-antibody complexes were eluted by incubation with a buffer containing 50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3 at 37°C for 15 minutes. IP samples and input control were treated with RNase A added to a final concentration of 10 µg/ml for one hour at 37°C. Then Proteinase K (Genaxxon Bioscience M3037) and NaCl were added to final concentrations of 80 µg/ml and 200 mM, respectively and samples were incubated over night at 65°C for protein digestion and reversal of crosslinks. DNA was precipitated and the input samples were used to assess the fragment size by gel electrophoresis on a 2% agarose E-Gel and on a DNA1000 bioanalyzer ChIP. Experiments were conducted for two replicates of each cell line.
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| Contributor(s) |
Thorn GJ, Clarkson CT, Mamayusupova H, Rademacher A, Schotta G, Rippe K, Teif VB |
| Citation(s) |
35387992 |
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| Submission date |
Sep 29, 2020 |
| Last update date |
Apr 27, 2022 |
| Contact name |
Vladimir B Teif |
| E-mail(s) |
vteif@essex.ac.uk
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| Organization name |
University of Essex
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| Department |
School of Life Sciences
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| Street address |
Wivenhoe Park
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| City |
Colchester |
| ZIP/Postal code |
CO4 3SQ |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA666407 |
| SRA |
SRP285796 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE158744_Atrx_peaks.bed.gz |
111.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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