Fig. 6. Models of chromatin assembly and structural organization on input alphoid BACs. Chromatin assemblies and transcripts on one unit of multimerized alphoid BAC DNA (7C5-SV BAC in A, 7C5-SV/CMV BAC in B–D) are shown as hypothetical models. Chromatin states supported by RT-PCR and ChIP analyses are indicated by colored lines. Arrows below the vector maps show transcription level (width) and length. Even though the ChIP analysis represents the sum of chromatin structures formed on multimerized alphoid BAC units, our ChIP data show a tendency for distinct chromatin structures to correspond to the sequence structures of the alphoid BAC constructs, implying that the HAC is maintained as punctuated blocks of chromatin structures. (A) Open chromatin or euchromatin (green) at the transcriptional gene on the right arm enhances the assembly of centromere chromatin (red) on the inserted alphoid DNA. In addition, when heterochromatin assembly (blue) occurs at the left arm and at a part of alphoid repeats (Fig. 4), functional HAC is generated and stably maintained. (B) If the heterochromatin-formation domain on the left arm is replaced with euchromatin by inserting a transcriptional gene unit, centromere-kinetochore components still can assemble on the alphoid array, but cohesion and inner centromere functions are absent. As a result, the unstable structure of the extra-chromosome is lost or integrated into a host chromosome. (C) Otherwise, at the integration site of a host chromosome, a silencing effect may be induced and/or heterochromatin spreads as indicated by triMet H3-K9 (Fig. 4A) into both non-selective marker gene and the insert alphoid DNA; consequently, centromere assembly on the alphoid DNA would be inactivated. (D) On the ectopic integration sites of multiple alphoid BAC DNAs, however, a chromatin opening also occurs stochastically in a part of the multiple array as indicated with the variegate assembly of the CENPs at the sites. If the open chromatin is selected again by the double selection as described in B, the chromatin opening and the functional assembly of kinetochore components on the ectopic alphoid array are accelerated and causes the reformation of minichromosomes accompanied by chromosome breakage events and with a part of the host chromosome fragment as a donor of heterochromatin-cohesion.