Epigenetic Characteristics and Gene Expression Analysis of a de novo Centromere in Maize

The eukaryotic centromere adopts a distinct chromatin state from euchromatin and heterochromatin, and varies greatly among different species. De novo centromeres originate occasionally from non-centromeric region of chromosomes, providing an excellent model system to study centromeric chromatin. The maize mini-chromosome Dp3a contains a de novo centromere, which was derived from a euchromatic site on the long arm of chromosome 3 that lacks traditional centromeric repeat sequences. Our previous study found that the CENH3 binding domain of this de novo centromere is only 350 kb with high-density gene distribution and low-density TE distribution, which was opposite from most neocentromeres in plants. Here we found a stable kinetochore complex assembled on the de novo centromere, indicating that this chromatin state resembles the native centromeres. Meiotic analyzes showed that the Dp3a mini-chromosome formed bi-orientation during meiosis I and could stably transmit through meiosis. We applied next generation sequence technology to analyze gene transcription, DNA methylation and histone modifications for this region. Our RNA-seq data revealed that active chromatin is not a barrier for centromere formation and transcription of genes in this region were suppressed after de novo centromere formation. The genes expression level within the Dp3a breakpoints of native chromosome 3 indicated a gene dosage effect exists due to additional copy of Dp3a mini-chromosome region in chromosome 3. Bisulfite-ChIP-seq results indicate a slightly increased DNA methylation level after de novo centromere formation, reaching the level of a native centromere, and no strand-specific bias of DNA methylation was found at maize native and Dp3a de novo centromeres. H2A-T133 phosphorylation occurs in the CENH3 nucleosome. All these results shed light on the mechanism of de novo centromere formation and subsequent consequences.