Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-A Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes.

BACKGROUND

The centromere is the specialized chromatin region that directs chromosome segregation. The kinetochore assembles on the centromere, attaching chromosomes to microtubules in mitosis. The centromere position is usually maintained through cell cycles and generations. However, new centromeres, known as neocentromeres, can occasionally form on ectopic regions when the original centromere is inactivated or lost due to chromosomal rearrangements. Centromere repositioning can occur during evolution. Moreover, de novo centromeres can form on exogenously transformed DNA in human cells at a low frequency, which then segregates faithfully as human artificial chromosomes (HACs). How centromeres are maintained, inactivated and activated is unclear. A conserved histone H3 variant, CENP-A, epigenetically marks functional centromeres, interspersing with H3. Several histone modifications enriched at centromeres are required for centromere function, but their role in new centromere formation is less clear. Studying the mechanism of new centromere formation has been challenging because these events are difficult to detect immediately, requiring weeks for HAC selection.

RESULTS

DNA injected into the Caenorhabditis elegans gonad can concatemerize to form artificial chromosomes (ACs) in embryos, which first undergo passive inheritance, but soon autonomously segregate within a few cell cycles, more rapidly and frequently than HACs. Using this in vivo model, we injected LacO repeats DNA, visualized ACs by expressing GFP::LacI, and monitored equal AC segregation in real time, which represents functional centromere formation. Histone H3K9 and H4 acetylations are enriched on new ACs when compared to endogenous chromosomes. By fusing histone deacetylase HDA-1 to GFP::LacI, we tethered HDA-1 to ACs specifically, reducing AC histone acetylations, reducing AC equal segregation frequency, and reducing initial kinetochroe protein CENP-A and NDC-80 deposition, indicating that histone acetylations facilitate efficient centromere establishment. Similarly, inhibition of RNA polymerase II-mediated transcription also delays initial CENP-A loading.

CONCLUSIONS

Acetylated histones on chromatin and transcription can create an open chromatin environment, enhancing nucleosome disassembly and assembly, and potentially contribute to centromere establishment. Alternatively, acetylation of soluble H4 may stimulate the initial deposition of CENP-A-H4 nucleosomes. Our findings shed light on the mechanism of de novo centromere activation.