Fig. 4. Identification of the CENP-C DNA-binding region in vivo. (A) Schematic
representation of the CENP-C mutants (left). The localization of the putative
DNA-binding domain (gray boxes) and the N-terminal HA tag (dark boxes) are
indicated. The right panel shows levels of protein expression detected by
western blot in HEK293T cells transfected with equal amounts of the mutant
constructs. 20 µg of total cell extracts were resolved on SDS-PAGE,
transferred onto a nitrocellulose membrane and probed either with anti-CENP-C
or anti-HA antibodies. (B) HEK293T cells were separately transfected with each
mutant construct and analyzed by ChIP assay. Chromatin was immunoprecipitated
either with anti-HA (IP anti-HA) or with anti-CENP-C antibodies (IP
anti-CENP-C). Immunoprecipitated DNAs along total DNA were hybridized with
alpha-satellite probes recognizing the centromeres of chromosomes 7, 13 and 21
(top left). The same filter was stripped and then hybridized with an Alu probe
(top right). A histogram representation of the relative enrichment of
alpha-satellite (bottom left) and Alu sequences (bottom right) obtained for
each mutants is shown. For each specific condition of immunoprecipitation (IP
anti-HA or IP anti-CENP-C), the enrichment (expressed in arbitrary units) is
defined as the ratio of the hybridization signal obtained for each sample
versus the hybridization signal obtained from cells transfected with the empty
vector and subjected to IP with the anti-HA antibody (IP background). The
standard error was calculated on the results of three independent experiments.
(C) Wild-type and truncated forms of the CENP-C protein are shown to bind the
same alpha-satellite subfamilies. Chromatin of HEK293T cells transfected with
the empty vector was immunoprecipitated either with anti-CENP-C or with
anti-HA antibodies. The mutant proteins were immunoprecipitated with anti-HA
antibodies. The analysis of the DNA profile was performed as described in
Fig. 2.
