Elucidating the localization of centromeric protein a (cenp-a) and the effect of germline and embryonic transcription on cenp-a localization in single cells in holocentric c. elegans

Abstract

Centromeres are the genomic locus that direct chromosome segregation during cell division. In most eukaryotes, centromere is defined epigenetically by the presence of the centromere-specific histone H3 variant CENP-A, interspaced with H3 nucleosomes. CENP-A nucleates assembly of the kinetochore, a multi-protein complex that binds to microtubules to orchestrate chromosome movement. In C. elegans holocentric chromosomes, centromeric nucleosomes are not occupying the whole genome, but only the poleward-facing mitotic chromatin, as a band. The 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 nuclei shows that CENP-A is only available to occupy at most 4% of the genome in individual cells. The exact, fewer locations of CENP-A binding in single cells may have been occluded by large population chIP studies. Previous study also show that CENP-A generally anti-correlates with germline and embryonic transcription based on population CENP-A and RNA Pol II chIP-chip and RNA-seq analyses. However, some CENP-A-enriched sites do exhibit transcription in late embryonic stages, while some germline-only or early embryonic gene regions do not have CENP-A binding throughout embryonic development. These findings complicate the understanding of the relationship between transcription and CENP-A localization. To delineate the exact pattern of CENP-A binding in single cell, we have constructed a transgenic DNA methylation enzyme fused with CENP-A to analyze methylated DNA marks in proximity to CENP-A binding in individual cell (DamID). This approach will reduce the averaging effects and enable us to examine the cell-to-cell variation, and variations due to developmental stages and transcription pattern changes.