Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks—deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

Toxoplasma gondii UBL-UBA Shuttle Protein DSK2s Are Important for Parasite Intracellular Replication

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin–proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis . These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation .

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, supporting a biological function in gene expression regulation.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis . These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using the published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. In total, our results indicate sisRNAs are selected intron regions with distinct properties, supporting a biological function in gene expression regulation.

Re-configuration of Chromatin Structure During the Mitosis-G1 Phase Transition

Higher-order chromatin organization such as A/B compartments, TADs and chromatin loops are temporarily disrupted during mitosis. These structures are thought to organize aspects of gene regulation, and thus it is important to understand how they are re-established after mitosis. We examined the dynamics of chromosome reorganization by Hi-C at defined time points following exit from mitosis in highly purified, synchronous cell populations. We observed that A/B compartments are rapidly established and progressively gain in strength following mitotic exit. Contact domain formation occurs from the “bottom-up” with smaller sub-TADs forming initially, followed by convergence into multi-domain TAD structures. CTCF is strongly retained at a significant fraction of sites on mitotic chromosomes and immediately resumes full binding at ana/telophase, the earliest tested time point. In contrast, cohesin is completely evicted from mitotic chromosomes and resumes focal binding with delayed kinetics. The formation of CTCF/cohesin co-anchored structural loops follows the kinetics of cohesin positioning. Stripe-shaped contacts anchored by CTCF grow in length, consistent with a loop extrusion process after mitosis. Interactions between cis-regulatory elements can form rapidly, preceding CTCF/cohesin anchored structural loops. Strikingly, we identified a group of rapidly emerging transient contacts between cis-regulatory elements in ana/telophase, that are dissolved upon G1 entry, co-incident with the establishment of inner boundaries or nearby interfering loops. Our findings indicate that distinct but mutually influential forces drive post-mitotic chromatin re-configuration to shape compartments, contact domains, cis-element contacts, and CTCF/cohesin dependent loops.