Fig. 1.

Tethering of p65 and VP16 into the alphoid^tetO HAC kinetochore creates two distinct types of ‘open’ chromatin. (A) Schematic drawings of the alphoid^tetO DNA array and tetR fusion constructs expressed in HeLa 1C7 cells. The synthetic monomer was based on a published consensus sequence for alphoid DNA (Choo et al., 1991). (B) Real-time RT-PCR analysis of non-transfected 1C7 cells stably carrying the alphoid^tetO HAC and 2 days after transfecting constructs expressing tetR-EYFP (tetR), tetR–EYFP–p65 (p65) or tetR–EYFP–VP16 (VP16). We selected for transfected cells expressing the tethered p65 and VP16 constructs using an IRES-Puro vector by puromycin selection. Expression levels of the alphoid^tetO array (tetO) and the chromosome 21 centromere (chr. 21) were normalized to those of β-actin. Alphoid^tetO RNA levels in untransfected cells were arbitrarily set as 1.0. Data represent the mean and s.e.m. of three or more independent experiments. (C) ChIP analysis of nontransfected 1C7 cells or cells transfected as in B using an antibody against the elongating form of RNA polymerase II. Transfections with tetR–EYFP–VP16 were performed either in the absence of or in the presence (+Dox) of 1 μg/ml doxycycline, which prevents the binding of tetR to the alphoid^tetO array. RNA polymerase II association with alphoid^tetO (tetO), chromosome 21 alphoid (chr. 21) DNA, the blasticidin S resistance (BSr) marker in the BAC vector and the endogenous PABPC1 gene was determined. Values were normalized to the RNA polymerase II occupancy at the endogenous PP1A housekeeping gene locus. Data represent the mean and s.e.m. of two or more independent experiments.