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CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains

Valeria Politi^1,*, Giovanni Perini^1,*, Stefania Trazzi¹, Artem Pliss^2,3, Ivan Raska^2,3, William C. Earnshaw⁴ and Giuliano Della Valle^1,

¹ Department of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy
² Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, CZ-12800 Prague 2, Czech Republic
³ Laboratory of Gene Expression, 1st Faculty of Medicine, Charles University, Albertov 4, CZ-12800 Prague 2, Czech Republic
⁴ Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Boulevard, Mayfield Road, Edinburgh EH9 3JR, Scotland
^* These authors contributed equally to this work

View larger version (27K): [in a new window]   Fig. 1. CENP-C binds the alpha-satellite DNA in vivo. (A) HeLa cells were formaldehyde treated and chromatin was immunoprecipitated (Formaldehyde-ChIP) with the different sera. After reversal of crosslinking, the immunoprecipitated DNA samples were analyzed by dot blot. The level of enrichment of different human highly repetitive sequences is shown (left). Decreasing amounts of total DNA are included to provide an internal quantitation of the hybridization signals (right). The filters were sequentially hybridized with the following [-32P]-labeled DNA probes: pZ7.6B (680 bp) and pZ21.A(850 bp) alpha-satellite DNAs, specific for chromosomes 7, 13 and 21; Sau3 A repeat (291 bp) and Alu repeat (300 bp). PI serum (rabbit preimmune serum), anti-CENP-B, anti-CENP-C and anti-ScII indicate the DNA recovered with the corresponding antibodies. (B) Densitometric quantitation of the hybridization signals. The intensity of the hybridization signal in the pre-immune samples was set to 1 arbitrary unit. (C) Level of enrichment of a single copy sequence (renin promoter region 350 bp) tested by semiquantitative PCR in the different immunoprecipitated samples. For each sample, three conditions of PCR (30-35-40 cycles) were used. Lanes 1-3, input (1 ng); lanes 4-6, PI serum; lanes 7-9, rabbit anti-CENP-B serum; lanes 10-12, rabbit anti-CENP-C serum. (D) HeLa cells were exposed to UV light for 15 minutes, lysed and chromatin was immunoprecipitated with the different sera. Immunoprecipitated DNA samples were hybridized with a cocktail of the pZ76.B and pZ21.A probes.

View larger version (27K): [in a new window]   Fig. 2. CENP-B and CENP-C bind the alpha-satellite DNA of different human chromosomes and recognize the same subfamilies of DNA sequences. (A) Filters were prepared as described in Fig. 1A and sequentially hybridized with alpha satellite probes recognizing, at high stringency, only a subset of human centromeric sequences. The specific chromosomes recognized by each probe are indicated. PI serum (pre-immune serum), anti-CENP-B and anti-CENP-C indicate the DNA recovered with the corresponding antibodies. (B) HeLa cells were formaldehyde-treated and chromatin was immunoprecipitated with the different sera. After reversal of crosslinking, DNA was digested with EcoRI, run on a 1.5% agarose gel and then blotted onto nylon membrane. Alpha-satellite DNA was revealed by hybridizing the filter with probes specific for alpha-satellite of chromosomes 7, 13 and 21. Dimers, tetramers and pentamers are multimers of the 171 bp alpha-satellite monomeric unit.

View larger version (48K): [in a new window]   Fig. 3. CENP-C recognizes DNA within the alpha-satellite arrays. Immunoprecipitated DNA samples were digested with EcoRI either before (lanes marked with an asterisk) or after the reversal of crosslinking. Alpha-satellite DNA was detected as previously described. For the immunoprecipitation with anti-CENP-C antibodies, two exposure times of the filter are presented: 16 hours and 48 hours. The alpha-satellite immunoprecipitated with anti-CENP-C and anti-CENP-B antibodies show a very similar band profile when digested with EcoRI either before or after the reversal of crosslinking.

View larger version (25K): [in a new window]   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.

View larger version (41K): [in a new window]   Fig. 5. CENP-B and CENP-C define different alpha-satellite domains. Cell extracts from crosslinked cells were treated for immunoprecipitation with pre-immune serum (lane 2), anti-CENP-B (lane 3), anti-CENP-C (lane 4) or anti-ScII (lane 7) antibodies, or first immunodepleted for the DNA—CENP-B complexes and then subjected to immunoprecipitation with anti-CENP-C (lane 5). As a control, a sample immunodepleted with anti-CENP-B antibodies was subjected to a second round of immunoprecipitation with the same antibody to verify the complete depletion of the DNA—CENP-B complex (lane 6). Alpha-satellite DNA, digested with EcoRI, was detected as described in Fig. 2.

View larger version (53K): [in a new window]   Fig. 6. CENP-B and CENP-C localize to different centromere domains in interphase chromosomes. (A-F) Immunoelectron microscopy showing simultaneous colocalization of CENP-B (10 nm gold particles) and CENP-C (6 nm gold particles). Each micrograph shows the centromere of an unidentified chromosome from a single cell nucleus. (A'-F') The maximum perimeter occupied by the CENP-B gold particles is marked for illustrative purposes. CENP-C particles within this perimeter are marked in red, while those outside the perimeter are marked in green. The bar in panel F' represents 100 nm.