PgmNr C32: Genetic and epigenetic control of DNA replication in Tetrahymena thermophila.

Authors:
Geoffrey Kapler 1 ; Xiangzhou Meng 1 ; Chunxiao Ge 1 ; Yifan Liu 2 ; Shan Gao 3


Institutes
1) Texas A&M University, College Station, TX; 2) University of Michigan, Ann Arbor, MI; 3) Ocean University of China, Qingdao, China.


Abstract:

The 21 kb T. thermophila ribosomal DNA (rDNA) minichromosome initiates DNA replication once per cell cycle during vegetative growth from origins that reside in the 5’ non-transcribed spacer (5’ NTS). Using Q-PCR, we show that the rDNA is replicated by two distinct modes during macronuclear development- locus-specific gene amplification, and genome-wide endoreplication. 5’ NTS origins mediate the initial wave of replication- amplification from 2C to 800C in starved mating cultures. This occurs in a restricted temporal window, concurrent with genome-wide endoreplication of non-rDNA macronuclear chromosomes from 2C to 8C. Upon re-feeding, the rDNA is no longer selectively amplified. Instead, it is endoreplicated to ~9000C along with the remainder of the genome, which achieves a copy number of ~45C. ORC protein levels are reduced during the second wave of DNA synthesis and known origins in the rDNA 5’ NTS are frequently bypassed. Aberrantly migrating rDNA replication intermediates (RIs) form at this time; their migration in 2D gels is consistent with the accumulation of stable RNA-DNA hybrids in the 5’ NTS. These RIs are indistinguishable from intermediates generated in a vegetative TXR1 knockout mutant strain, defective in histone H3K27 monomethylation.

To explore the possible relationship between H3K27me1 and endoreplication, the average inter-origin distance (IOD) was determined by DNA fiber analysis. This genome-wide approach revealed significant differences in replication initiation in endoreplicating wild type cells and vegetative TXR1 mutants. Despite the down-regulation of Orc1p in endoreplicating mated/re-fed cells, IOD decreased. Paradoxically, more initiation events occur when ORC levels are reduced.  In contrast, the IOD increased in the vegetative TXR1 knockout mutant. Thus, although the formation of aberrant RIs is conserved, the mechanism underlying their biogenesis may differ. Finally, RNA-Seq revealed that TXR1 mRNA levels peaks in G1, prior to the onset of DNA replication. In situ immunofluorescence confirmed cell cycle regulation at the protein level. We propose a model in which H3K27me1 facilitates the assembly of ORC-dependent pre-replicative complexes (pre-RCs) during G1 phase. We speculate that the failure to establish pre-RCs triggers an alternative DNA replication program that bypasses the requirement for ORC. A recently published study, in which vegetative Orc1p levels are transiently reduced 50-fold, is consistent with the existence of an alternative pathway for the initiation of DNA replication in Tetrahymena.