Darmon, E. | bioRxiv Science

bioRxiv2020

The transcription factor TRF2 has a unique function in regulating cell cycle and apoptosis

TRF2 (TATA-box-binding protein-related factor 2) is an evolutionarily conserved general transcription factor that is essential for embryonic development of Drosophila melanogaster, C. elegans, zebrafish and Xenopus. Nevertheless, the cellular processes that are regulated by TRF2 are largely underexplored. Here, using Drosophila Schneider cells as a model, we discovered that TRF2 regulates cell cycle progression. Using flow cytometry, high-throughput microscopy and advanced imaging-flow cytometry, we demonstrate that TRF2 knockdown regulates cell cycle progression and exerts distinct effects on G1 and specific mitotic phases. RNA-seq analysis revealed that TRF2 regulates the expression of Cyclin E and the mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, but not Cyclin D or Cyclin C. To identify proteins that could account for the observed regulation of these cyclin genes, we searched for TRF2-interacting proteins. Interestingly, mass spectrometry analysis of TRF2-containing complexes identified GFZF, a nuclear glutathione S-transferase implicated in cell cycle regulation, and Motif 1 binding protein (M1BP). Furthermore, available ChIP-exo data revealed that TRF2, GFZF and M1BP co-occupy the promoters of TRF2-regulated genes. Using RNAi to knockdown the expression of either M1BP, GFZF, TRF2 or their combinations, we demonstrate that although GFZF and M1BP interact with TRF2, it is TRF2, rather than GFZF or M1BP, that is the main factor regulating the expression of Cyclin E and the mitotic cyclins. Taken together, our findings uncover a critical and unanticipated role of a general transcription factor as a key regulator of cell cycle.

molecular biology↗

bioRxiv2019

DNA Double Strand Break Repair in E. coli Perturbs Cell Division and Chromosome Dynamics

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.\n\nAuthor SummaryThe bacterium Escherichia coli has a remarkable cell cycle where overlapping rounds of DNA replication can occur in a single generation between cell birth and division. This implies a complex coordination network between growth, genome duplication and cell division to ensure that the right number of genomes are created and distributed to daughter cells at all growth rates. This network must be robust to a number of unpredictable challenges. One such challenge is broken DNA, something that in E. coli is estimated to occur in ~20% of cell division cycles. In this work we perturb the E. coli cell cycle by elevating the frequency of repairable DNA double-strand breaks to determine which parameters of the cell cycle are conserved and which are changed. Our results demonstrate that this perturbation does not alter the average cell size at initiation of DNA replication or initiation of cell division. Furthermore, it does not alter the time taken to replicate the genome or the generation time. However, it does delay the segregation of the DNA to daughter cells and the completion of cell division explaining the increase in average cell size observed previously.

genetics↗