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. 2008 Mar 31;28(11):3573–3588. doi: 10.1128/MCB.00087-08

FIG. 7.

FIG. 7.

ChIP assays for factors bound to U2 genes at interphase and metaphase. (A) SNAPc and Pol II CTD phosphorylation on the U2 snRNA promoter (−264 to +15) and downstream flanking sequence (+791 to +1019) in HT1080 cells. The abundance of SNAPc on the promoter compared to the downstream fragment demonstrates that ChIP has sufficient resolution for these experiments, although chromatin shearing to the limit size of 500 bp leaves many fragments of >2 kb which reduce resolution on the small U snRNA genes. (B) SNAPc lost from the U2 promoter at metaphase in HT1080 cells. Quantification below ChIP panels is the ratio of mitotic to asynchronous signals when normalized to 50 ng input (upper row) or 1% input DNA (lower row). (C) Some SNAPc 43 but little SNAPc 190 remains on the U2 promoter in the CSB null cell line GM00739Bhtert, derived from patient CS1AN. Quantification is as in panel B. (D) Some SNAPc 43 but little SNAPc 190 remains on the U2 promoter at metaphase in HT1080 cells treated with a low concentration of actinomycin D to induce RNU2 fragility. Quantification is as in panel B. (E) Most SNAPc 43 but little SNAPc 190 remains on the U2 promoter at metaphase in HT1080 cells transduced with the tetrameric C-terminal domain of p53 (TAT-p53CTD) to induce RNU2 fragility. Quantification is as in panel B. (F) Neither SNAPc 43 nor SNAPc 190 is retained at metaphase on the promoter of the largest subunit of RNA polymerase II in untreated HT1080 cells, HT1080 treated with actinomycin D or transduced with TAT-p53CTD, or in mitotic GM00739Bhtert or WI38htert cells. (G) CSB can be detected on active rRNA transcription units (PCR primers amplify +3639 to +3798), but only the CSB N terminus is accessible to our antibody. (H) Neither CSB nor BRCA1 can be detected on the U2 snRNA promoter before or after treatment of asynchronous HT1080 cells with actinomycin D. Pisces is a polyclonal antibody directed against the C terminus of CSB, and Jasmine is a polyclonal antibody against the N terminus (80); “comm” is a commercial polyclonal antibody against CSB (Santa Cruz).