Titration of Four Replication Factors Is Essential for the Xenopus laevis Midblastula Transition

Abstract

The rapid, reductive early divisions of many metazoan embryos are followed by the mid-blastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the mid-blastula transition (MBT). We show that four DNA replication factors, Cut5, RecQ4, Treslin, and Drf1, are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis . The levels of these factors regulate multiple events of the MBT including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.The early cell divisions in many metazoa are rapid and maintained by maternally supplied products. Bulk zygotic transcription begins at the mid-blastula transition (MBT), also referred to as the start of the maternal-zygotic transition (MZT) (1), and is accompanied by the onset of cell movement, the activation of cell cycle checkpoints and the elongation and asynchrony of cell divisions (2-6). This elongation of the cell cycle is coincident with a reduction in the density and synchrony of DNA replication initiation events (7-9), leading to the possibility that replication factors themselves might be important regulators of cell cycle duration during development. To test this hypothesis we measured the abundance of replication initiation factors in Xenopus laevis embryos. Components of the first step of replication initiation, a process called 'licensing' or pre-replicative complex (pre-RC) assembly, remained constant or increased in abundance during embryogenesis ( After pre-RC formation, two kinases are required to assemble active replisomes, CyclinDependent Kinase (CDK) and Dbf4-Dependent kinase (DDK). The abundance of the noncatalytic subunit of DDK, which in Xenopus embryos is Drf1 (Dbf4-related factor 1), becomes greatly reduced by the time of gastrulation (10, 11). Both the mRNA and protein levels of three other replication factors, cut5, treslin and recq4 also decreased during embryogenesis ( Fig. 1A and Fig. S1). (12)(13)(14). Reduction in the levels of these replication factors during early cell divisions suggested that their abundance might be a determinant of embryonic replication initiation rates. To test this, we added in vitro translated (IVT) RecQ4, Treslin, Cut5, and Drf1 to Xenopus egg extracts and analyzed the replication of sperm nuclei in vitro. Increased expression of these proteins in extracts resulted in a corresponding increase in DNA synthesis when these factors were overexpressed in combination ( Fig. S2A-E). Furthermore, increasing the nuclei:extract ratio, to mimic the increasing N/C ratio of the early embryo, caused an exponential decline in DNA synthesis, which was suppressed by overexpression of RecQ4, Treslin,...

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

“…In contrast, the TICRR TESE phosphomimetic mutant can shorten S phase. Our results, consistent with the work of Collart et al (2013), support a model in which S-phase length can be controlled through TICRR phosphorylation, and the important effect of TICRR phosphorylation is to promote the formation of TICRR-TopBP1 complexes (Kumagai et al 2011). Notably, TICRR TESE overexpression can suppress the effects of CDK2 inhibition but not DDK inhibition.…”

“…In yeast and X. laevis, homologs of TICRR are among several initiation factors that are limiting for replication initiation (Mantiero et al 2011;Collart et al 2013). TICRR is also phosphorylated by CDK on two residues (T969 and S1001), and CDK is limiting for S-phase progression (Boos et al 2011;Kumagai et al 2011).…”

“…In X. laevis, a sharp extension in S-phase length occurs during early embryonic development, and, at the same time, the level of four proteins necessary for DDK and CDK signaling are reduced (Collart et al 2013). This suggests that S-phase lengthening is caused by the reduced expression of these four factors.…”

Evolutionary diversification ofMCM3genes inXenopus laevisandDanio rerio

“…13 Furthermore, a recent study suggests that limitation of the number of activators of the pre-RC plays a key role in stopping the embryonic cell cycle and entering MBT. 14 This implies that quantitative differences in replication proteins contribute to the different replication programs found in embryonic vs. somatic cell cycles. But it remains to be clarified whether qualitative differences of replication proteins also contribute to the program.…”

Cdc25 and the importance of G2control

“…14,27 Instead, Chk1 activation depends on the depletion of specific DNA replication factors and limiting dNTP levels, which occurs at a precise nucleocytoplasmic ratio. 29 Although we now know quite a bit about the mechanism of the MBT in select model organisms, why does an MBT even exist? By executing a very fast cell cycle without transcription, embryos can quickly reach a multi-cellular stage.…”