Epigenetic origin of evolutionary novel centromeres

Abstract

Most evolutionary new centromeres (ENC) are composed of large arrays of satellite DNA and surrounded by segmental duplications. However, the hypothesis is that ENCs are seeded in an anonymous sequence and only over time have acquired the complexity of "normal" centromeres. Up to now evidence to test this hypothesis was lacking. We recently discovered that the well-known polymorphism of orangutan chromosome 12 was due to the presence of an ENC. We sequenced the genome of an orangutan homozygous for the ENC, and we focused our analysis on the comparison of the ENC domain with respect to its wild type counterpart. No significant variations were found. This finding is the first clear evidence that ENC seedings are epigenetic in nature. The compaction of the ENC domain was found significantly higher than the corresponding WT region and, interestingly, the expression of the only gene embedded in the region was significantly repressed.

Figure 1. Immuno-FISH characterization of the orangutan ENC12.

(a) The metaphase is from PPY-10, heterozygous for the ENC12, hybridized with a pool of alphoid centromeric sequences obtained as described in Methods (red signal). The short arrow indicates the normal PPY12, while the long one points to the ENC12 chromosome. (b) The partial metaphase is from the PPY-15 homozygous. The two ENC12 chromosomes (arrowed) show the FISH signal of the centromeric alphoid sequences (red) at the old deactivated centromere and the immuno signal (green) at the functional ENC12 centromere. The immuno signal was obtained using antibodies against the centromeric protein CENP-C.

Figure 2. ENC12 CHIP-on-chip results and mapping of the sequenced BACs.

The Figure graphically reports the ChIP-on-chip results. DNA obtained by chromatin immunoprecipitation, using an anti-CENP-A antibody, was hybridized to a tiling array covering the neocentromeric region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA. The Figure also reports the position of the five CH276 BAC clones that were PacBio sequenced (see the following paragraphs), with respect to the ponAbe2 sequence, and to the ChIP-on-chip results.

Figure 3. Sequence comparison of the ENC12 domain versus WT.

Dot plot matrix comparing PacBio805 sequence (Y axis) to the corresponding region in NGS12 sequence (ENC12, X axis), using Gepard-1.40. Sequence lengths are given at the axis ends.

Figure 4. ENC chromatin compaction.

(a) Example of an immuno-FISH experiment on an interphase nucleus of the heterozygous PPY-10 individual. The compaction was measured using the BACs CH276-136P13 (red signal) and CH276-12M5 (blue signal), flanking the ENC12. Their precise position on the ponAbe2 assembly is reported in the Fig. 2. The green immuno signal of CENP-C was used to discriminate the ENC12 domain (long arrow) from the WT counterpart (short arrow). (b) The boxplot shows the distributions of interprobe normalized distances for the ENC12 and its WT counterpart. P-value was calculated by Mann-Whitney U-tests.