Journal: bioRxiv

Article Title: Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation

doi: 10.1101/407031

Figure Lengend Snippet: a) mESCs expressing rTetR-Dam-EGFP-ERT2 allow to control the nuclear concentration of the fusion protein by changing the amount of 4-hydroxy-tamoxifen (4-OHT) in the culture medium. b) Nuclear concentration of the rTetR-Dam fusion protein as a function of 4-OHT concentration. Number of protein copies per nucleus were determined using mass spectrometry on nuclear extracts and divided by the average nuclear volume (∼490 fl) determined using DAPI staining (see ). Error bars corresponds to the standard deviation of the two experimental replicates. c) Random integration of large numbers of 50x TetO platforms using the piggyBac transposon. Accumulation of EGFP signal to nuclear foci in the presence of Dox (right: max. intensity projection over 10 Z planes) indicates binding of rTetR-Dam to the arrays and allows selecting clones with large numbers of insertions. d) Quantification of damC experiments as a function of rTetR-Dam concentration. damC enrichment in the immediate vicinity of TetO viewpoints (contact probability ∼1) shows a maximum in line with the model prediction. Blue: Average +/- standard deviation over the 100 TetO viewpoints with highest enrichment. Red line: model fit to the experimental data.

Article Snippet: 5×10 6 cells were resuspended in 100 µl transfection solution (82ul primary solution, 18ul supplement 1, 2μg pBroad3-rTetR-EGFP-Dam-ERt2) and transferred in a single Nucleocuvette (Lonza).

Techniques: Expressing, Concentration Assay, Mass Spectrometry, Staining, Standard Deviation, Binding Assay, Clone Assay

Journal: bioRxiv

Article Title: Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation

doi: 10.1101/407031

Figure Lengend Snippet: a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see ). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler mass spectrometry . We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy34. Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the damC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´ → 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The damC sequencing library preparation protocol includes UMIs allowing to filter ∼15% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID30 at the same sequencing depth. f) Median DamC enrichment at 100 viewpoints with highest enrichment as a function of 4-OHT concentration. Significant amounts of damC enrichment can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5-10 nM 4-OHT in our experimental system.

Article Snippet: 5×10 6 cells were resuspended in 100 µl transfection solution (82ul primary solution, 18ul supplement 1, 2μg pBroad3-rTetR-EGFP-Dam-ERt2) and transferred in a single Nucleocuvette (Lonza).

Techniques: Concentration Assay, Binding Assay, Mass Spectrometry, Staining, Expressing, Ligation, Amplification, Next-Generation Sequencing, Sequencing, Methylation, Genome Wide