Genome-Wide Analysis of DNA Replication Origins in Tetrahymena Thermophila

Abstract

As a prototypic ciliated protozoan, Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During the vegetative phase of the life cycle, the S phases of the diploid germline micronucleus and polyploid somatic macronucleus are temporally offset. Here we report the first RNA-Seq analysis across the cell cycle of a bi-nucleated ciliated protozoan. RNA was isolated at 30 min intervals across 1.5 vegetative cell cycles, starting with a G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the predicted genes were shown to be cell cycle-regulated. Protein-coding genes that are shared between mics and MACs exhibit a single cell cycle mRNA peak, while the expression of nuclear-specific protein-coding genes peaks prior to their respective nuclear function. Cyclin D and Cyclin A/B genes showed two distinct patterns corresponding to two nuclei. Clustering of periodically expressed genes identified 7 gene expression clusters. Four of them have known biological process overrepresented corresponding to different phase of cell cycle. We propose that these clusters contain known and novel factors that coordinate mic- and mac-specific events.

Despite intense biochemistry and genetic investigation of rDNA replication and regulatory factors, Tetrahymena macronuclear replication origins remain elusive. Here we mapped Tetrahymena macronuclear replication origins on a genome-wide, for the first time, using two methods: Okazaki fragments sequencing (OK-seq) and nanopore sequencing (FORK-seq). We show that Tetrahymena replication initiated in compact zones around 5 kb often circumscribed by transcribed genes in non-transcribed regions downstream of protein-coding genes in macronuclei of vegetative growing cells. Replication origins are likely to have a strong sequence specific component, since like S. cerevisiae, we found conserved, poly (dA) and poly (dT) containing motifs on opposite strands that coincide or in close proximity to replication initiation zones. These motif are complementary to the 26T RNA subunit, present in all T. thermophila ORC ribonucleoprotein complexes. Beside sequence determinants, replication initiation zone are shaped by active transcription. Replication initiation sites TXR1 deletion strain were also studied compare to wild type using OK-seq. We showed the impact of H3K27me1 deficiency on replication initiation sites, and potential mechanisms for how H3K27me1 coordinates transcription and replication fork elongation to prevent head-on collisions.