Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
We used the recombineering technology (Liu, P., Jenkins, N.A. and Copeland, N.G. (2003) Genome Res.) to generate the targeting vectors that introduce the triple affinity tag in the subunits of Pol III, TFIIIB, TFIIIC, and TFIIS. The 46C ES cell line (Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003) Nat. Biotech.) was transfected by electroporation with each targeting vector. Cells were plated in D15 medium as described (Tessarollo, L. (2001). Methods Mol Biol.) and selected with G418. Individual ES cell colonies were collected seven days after electroporation, amplified and genotyped by Southern blotting, in order to identify the clones that underwent a homologous recombination event. In these clones, a sequence encoding a 6 Histidine-Flag-HA tag followed by a neomycin resistance marker flanked by loxP sites, was inserted just after the last codon of the gene encoding the protein to be tagged. For TCEA1, the tag was inserted just after the start codon. The integration of the cassette at the right loci was verified by Southern blotting using three or four different restriction enzymes and PCR. Transient transfection with a Cre recombinase expression vector was used to remove the selection cassette. The karyotypes of all cell lines were verified. The genes that were modified in the cell lines encoded RPC1 (MGI:2681836), RPC4 (MGI:1914315), BRF1 (MGI:1919558), BRF2 (MGI:1913903), TFIIIC220 (MGI:107887), TFIIIC110 (MGI:1919002), TFIIIC90 (MGI:2138937) and TCEA1 (MGI:1196624) respectively. The expression of the tagged versions of the proteins was verified by western blotting using HA7 anti-HA antibodies (Sigma). Untagged ES cell lines (46C, 46C-2) are used as a negative control for the ChIP-seq experiment. ES cells were cultured on embryonic fibroblast feeder cells blocked with mitomycin C in D15 medium (Dulbecco Modified Eagle Medium High Glucose supplemented with 15% FBS, 2mML-glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin, 0.1 mM nonessential amino acids (all from GIBCO), 0.1 mM 2-mercaptoethanol [Sigma] and 1,000 units/ml LIF). ES cells were maintained at 37C, 5% carbon dioxide, fed with fresh media daily, and transferred to new plates after trypsinization. Typically 150-200 million cells were collected for each ChIP experiment. The cellular proteins and DNA were cross-linked by the addition of formaldehyde (0.4% final concentration) in cell culture media. The plates were incubated for 10 min at room temperature then, the reaction was stopped by the addition of glycine (0.125 M final concentration). The cells were washed twice with 10 ml chilled PBS. Each plate was scraped with 2 ml PBS with protease inhibitors (Roche complete, 10 mM PMSF dissolved in ethanol), pelleted by centrifugation at 2500 rpm and resuspended in 1 ml per dish of FA/SDS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% Na-deoxycholate, 0.1% SDS) with protease inhibitors and incubated on ice for 15 min. The subsequent operations were performed at 4C. Chromatin was collected by centrifugation for 20 min at 12 000 rpm, resuspended in the same amount of FA/SDS and put on a rotating wheel for 1 hr, centrifuged and resuspended in 0.4 volume of FA/SDS with protease inhibitors. 400 ul batches of chromatin were fragmented with a sonicator (Diagenode) to generate DNA fragments of 300-400 bp mean size. The chromatin was collected by centrifugation at 14 000 rpm for 10 min and was kept at -80C until needed. The immunoglobulin-coupled magnetic beads (Dynal) used in the chromatin immunoprecipitation experiments were prepared essentially as described previously (Esnault, C., Ghavi-Helm, Y., Brun, S., Soutourina, J., Van Berkum, N., Boschiero, C., Holstege, F. and Werner, M. (2008) Mol. Cell). Anti-HA antibody was HA7 H-3663 (Sigma) and anti-Flag anti-body was F-1804 (Sigma). For each ChIP experiment, 240 ug DNA (RPC1, RPC4, 46C) or 750 ug DNA (BRF1, BRF2) of sonicated chromatin were incubated with 150 ul beads for 2 hr at room temperature. The beads were magnetically pelleted and the supernatant was removed. The beads were washed with 1 ml of low salt buffer (pH 8.0, 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), then with the same volume of high salt buffer (pH 8.0, 20 mM Tris-HCl, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), then with low LiCl buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate) and finally twice with TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The first ChIP was performed using the anti-HA antibody. The bound chromatin was eluted using FA/SDS buffer containing the HA peptide (0.5 mg/ml; Ansynth Service) for 4 h at 16C then overnight at 4C. For the second ChIP, the supernatant was incubated with 50 ul beads coupled to anti-Flag antibodies which were treated as above except for the elution that was performed by incubation with a buffer containing 1% SDS and 0.1 M NaHCO3. Reversal of the cross-links and DNA purification were performed as described previously (Kuras, L., Borggrefe, T. and Kornberg, R.D. (2003) Proc. Natl. Acad. Sci. USA).
Experimental Factor: IMMUNOPRECIPITATE
Chromatin IP against BRF1
Experimental Factor: GENOTYPE
BRF1 tagged