The BBB neocentromere contains a major and a minor centromere chromatin domain

DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-A, CENP-C, and CENP-H from cell line BBB was hybridized to a custom made microarray containing 257 unique polymerase chain reaction (PCR) fragments. Three independent biological replicates were performed for each antibody, and the scale normalized mean logCy-5:Cy-3 intensity ratios (ChIP to input), were plotted on the y-axis with the standard error (SE) for each PCR fragment. Intensity ratios at least three times the standard deviation (SD) from the background mean (dashed line) were considered positives (see Materials and methods). An alpha satellite containing plasmid was included as a positive control (far right). Centromere protein (CENP)-A ChIP. The major CENP-A domain was about 80.3 kilobases (kb; shaded region), with positive intensity ratios 1.17 to 2.46. The minor domain was about 8.5 kb (shaded region) and was approximately 162 kb downstream from the major domain; intensity ratios were 1.14 to 1.33. Background experimental mean was -0.39 ± 0.47 SD, one-tailed distribution cut-off was ≤ 0.68, positive values were ≥ 1.02 (dashed line). Alpha satellite = 1.63 ± 0.18 SE. CENP-C ChIP. Major CENP-C domain was 87.8 kb (shaded region). Intensity ratios were 0.67 to 3.41. Minor domain was 8.5 kb; intensity ratios were 0.65 to 1.07 (shaded region). Background experimental mean was -0.37 ± 0.34 SD, one-tailed distribution cut-off was ≤ 0.31, positive values were ≥ 0.65 (dashed line). Alpha satellite = 2.36 ± 0.70 SE. CENP-H ChIP. Major CENP-H domain was about 86.3 kb (shaded region), and positive intensity ratios were 0.64 to 3.35. Minor domain was about 1.9 kb (shaded region), and intensity ratios were 0.82 and 1.14. Background experimental mean was -0.33 ± 0.32 SD, one-tailed distribution cutoff was ≤ 0.56, positive values were ≥ 0.63 (dashed lines). Alpha sat = 2.06 ± 0.59 SE. The 2.3 megabase (Mb) region included in the PCR CHIP. The central 350 kb region, covered by PCR fragments at high density. The adjacent megabase on either side of the central region, shown at a 10 fold reduced scale, was covered by PCR fragments at decreasing density. PCR microarray fragments listed in Table 1, found at the edges of CENP-A, CENP-C and CENP-H domains, and the negative values within the first domain, are shown. The major and minor chromatin domains are shown by the rectangles. The tiling path of the unique sequenced regions of each bacterial artificial chromosome (BAC) and their overlaps are shown within the 350 kb region. The corresponding Repeat Masker data from the Human Genome Browser at UCSC and thegenes in the area are indicated [50].<p><b>Copyright information:</b></p><p>Taken from "Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres"</p><p>http://genomebiology.com/2007/8/7/R148</p><p>Genome Biology 2007;8(7):R148-R148.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC2323242.</p><p>