An additional cell cycle checkpoint: Cell division is controlled by checkpoints that regulate the temporal order of the cell cycle phases, including the G ₁ /S, G ₂ /M, and metaphase/anaphase transitions. Yet there are no known control mechanisms for a fourth fundamental transition—the S/G ₂ transition. Saldivar et al. report a switchlike control mechanism that regulates the S/G ₂ transition. The checkpoint kinase ATR senses ongoing DNA replication in S phase and represses the mitotic transcriptional network, ensuring that DNA replication in S phase is completed before mitosis. Science , this issue p. 806

Our understanding of the structure and function of mitotic chromosomes has come a long way since these iconic objects were first recognized more than 140 years ago, though many details remain to be elucidated. In this chapter, we start with the early history of chromosome studies and then describe the path that led to our current understanding of the formation and structure of mitotic chromosomes. We also discuss some of the remaining questions. It is now well established that each mitotic chromatid consists of a central organizing region containing a so-called "chromosome scaffold" from which loops of DNA project radially. Only a few key non-histone proteins and protein complexes are required to form the chromosome: topoisomerase IIα, cohesin, condensin I and condensin II, and the chromokinesin KIF4A. These proteins are concentrated along the axis of the chromatid. Condensins I and II are primarily responsible for shaping the chromosome and the scaffold, and they produce the loops of DNA by an ATP-dependent process known as loop extrusion. Modelling of Hi-C data suggests that condensin II adopts a spiral staircase arrangement with an extruded loop extending out from each step in a roughly helical pattern. Condensin I then forms loops nested within these larger condensin II loops, thereby giving rise to the final compaction of the mitotic chromosome in a process that requires Topo IIα.

Ki-67 is one of the most famous marker proteins used by histologists to identify proliferating cells. Indeed, over 30 000 articles referring to Ki-67 are listed on PubMed. Here, we review some of the current literature regarding the protein. Despite its clinical importance, our knowledge of the molecular biology and biochemistry of Ki-67 is far from complete, and its exact molecular function(s) remain enigmatic. Furthermore, reports describing Ki-67 function are often contradictory, and it has only recently become clear that this proliferation marker is itself dispensable for cell proliferation. We discuss the unusual organization of the protein and its mRNA and how they relate to various models for its function. In particular, we focus on ways in which the intrinsically disordered structure of Ki-67 might aid in the assembly of the still-mysterious mitotic chromosome periphery compartment by controlling liquid–liquid phase separation of nucleolar proteins and RNAs.

We used Hi-C, imaging, proteomics, and polymer modeling to define rules of engagement for SMC (structural maintenance of chromosomes) complexes as cells refold interphase chromatin into rod-shaped mitotic chromosomes. First, condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. Second, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion as sisters separate. Studies of mitotic chromosomes formed by cohesin, condensin II, and condensin I alone or in combination lead to refined models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase, loops are extruded in vivo at ∼1 to 3 kilobases per second by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

Background: In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. In the present study, we evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. In addition, we analyzed the association of CTCF and covalent histone modifications at the miR-125b1 locus. Methods: To assess the DNA methylation status of the miR-125b1, genomic DNA was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. The miR-125b1 gene expression was analyzed by qRT-PCR using U6 as a control for constitutive gene expression. CTCF repressive histone marks abundance was evaluated by chromatin immunoprecipitation assays. Results: The disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors. Conclusions: A reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoid^tetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres. This article has an associated First Person interview with the first authors of the paper.