Epigenetic regulation of centromere establishment in Caenorhabditis elegans and holocentric but not everywhere—Determine the localization of centromeric protein A on the genome by single cell DamID

Abstract

The centromere is a chromosomal region specialized for directing chromosome segregation. Due to the crucial function of the centromere, the existing centromere is usually maintained stably at the same position through mitotic 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, and even on exogenously introduced DNA. However, how centromeres are maintained, inactivated and activated is still unclear. Injecting DNA into the germline of the C. elegans leads to formation of artificial chromosomes (ACs) in embryos, which first undergo passive inheritance, but form centromeres and get the ability to autonomously segregate within a few cell cycles, much more efficiently than in human cells. Using this in vivo model, our results indicate that histone acetylations on chromatin and transcription can create an open chromatin environment that favors centromere establishment. Different histone modifications will be tested systematically in future, and this will give insights into the “histone code” required for de novo centromere formation. In holocentric chromosomes, centromeric nucleosomes are not occupying the whole genome, but only the poleward-facing mitotic chromatin, as a band. However, what is the CENP-A binding pattern in individual cells? Our cross-linked chromatin immunoprecipitation analysis from millions of embryo population shows that CENP-A is enriched in 50% of the genome, but CENP-A protein quantification in individual cells shows that CENP-A is only available to occupy at most 4% of the genome. The discrepancies in this conclusion may be due to the exact locations of CENP-A binding in single cells have been occluded by large population chIP studies. To resolve these discrepancies and to delineate the exact pattern of CENP-A binding in single cell, we will use a transgenic DNA methylation enzyme fused with CENP-A to elucidate the CENP-A binding pattern through methylated DNA marks in individual cell, to reduce the averaging effects in prior studies and examine the preference of binding, the cell-to-cell variation, variation due to developmental stages, and potential plasticity in CENP-A binding on holocentromeres.