Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle Progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.

Sowing the Seeds of Centromeres

The centromere is a chromatin-based platform that accumulates microtubule-binding proteins that drive chromosome segregation during cell division. Despite their size (on the order of megabases of DNA in mammals) and conserved role, centromeres have the remarkable capacity to leave their usual comfort zone and to reform at a new chromosomal site (1). Although found rarely, these so-called neocentromeres are by most measures bona fide and segregate chromosomes with high fidelity. What accounts for this nomadic behavior?

Temporal control of epigenetic centromere specification

All living organisms require accurate mechanisms to faithfully inherit their genetic material during cell division. The centromere is a unique locus on each chromosome that supports a multiprotein structure called the kinetochore. During mitosis, the kinetochore is responsible for connecting chromosomes to spindle microtubules, allowing faithful segregation of the duplicated genome. In most organisms, centromere position and function is not defined by the local DNA sequence context but rather by an epigenetic chromatin-based mechanism. Centromere protein A (CENP-A) is central to this process, as chromatin assembled from this histone H3 variant is essential for assembly of the centromere complex, as well as for its epigenetic maintenance. As a major determinant of centromere function, CENP-A assembly requires tight control, both in its specificity for the centromere and in timing of assembly. In the last few years, there have been several new insights into the molecular mechanism that allow this process to occur. We will review these here and discuss the general implications of the mechanism of cell cycle coupling of centromere inheritance.

Basic properties of epigenetic systems: lessons from the centromere

Chromatin-based epigenetic inheritance cooperates with cis-acting DNA sequence information to propagate gene expression states and chromosome architecture across cell division cycles. Histone proteins and their modifications are central components of epigenetic systems but how, and to what extent, they are propagated is a matter of continued debate. Centromeric nucleosomes, marked by the histone H3 variant CENP-A, are stable across mitotic divisions and are assembled in a locus specific and cell cycle controlled manner. The mechanism of inheritance of this unique chromatin domain has important implications for how general nucleosome transmission is controlled in space and time.

Premature Sister Chromatid Separation Is Poorly Detected by the Spindle Assembly Checkpoint as a Result of System-Level Feedback

Sister chromatid cohesion, mediated by the cohesin complex, is essential for faithful mitosis. Nevertheless, evidence suggests that the surveillance mechanism that governs mitotic fidelity, the spindle assembly checkpoint (SAC), is not robust enough to halt cell division when cohesion loss occurs prematurely. The mechanism behind this poor response is not properly understood. Using developing Drosophila brains, we show that full sister chromatid separation elicits a weak checkpoint response resulting in abnormal mitotic exit after a short delay. Quantitative live-cell imaging approaches combined with mathematical modeling indicate that weak SAC activation upon cohesion loss is caused by weak signal generation. This is further attenuated by several feedback loops in the mitotic signaling network. We propose that multiple feedback loops involving cyclin-dependent kinase 1 (Cdk1) gradually impair error-correction efficiency and accelerate mitotic exit upon premature loss of cohesion. Our findings explain how cohesion defects may escape SAC surveillance.

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

Novel insights into mitotic chromosome condensation

The fidelity of mitosis is essential for life, and successful completion of this process relies on drastic changes in chromosome organization at the onset of nuclear division. The mechanisms that govern chromosome compaction at every cell division cycle are still far from full comprehension, yet recent studies provide novel insights into this problem, challenging classical views on mitotic chromosome assembly. Here, we briefly introduce various models for chromosome assembly and known factors involved in the condensation process (e.g. condensin complexes and topoisomerase II). We will then focus on a few selected studies that have recently brought novel insights into the mysterious way chromosomes are condensed during nuclear division.

Centrioles: active players or passengers during mitosis?

No funding agencies or grants indicated in the publication.

Stepwise evolution of the centriole-assembly pathway

The centriole and basal body (CBB) structure nucleates cilia and flagella, and is an essential component of the centrosome, underlying eukaryotic microtubule-based motility, cell division and polarity. In recent years, components of the CBB-assembly machinery have been identified, but little is known about their regulation and evolution. Given the diversity of cellular contexts encountered in eukaryotes, but the remarkable conservation of CBB morphology, we asked whether general mechanistic principles could explain CBB assembly. We analysed the distribution of each component of the human CBB-assembly machinery across eukaryotes as a strategy to generate testable hypotheses. We found an evolutionarily cohesive and ancestral module, which we term UNIMOD and is defined by three components (SAS6, SAS4/CPAP and BLD10/CEP135), that correlates with the occurrence of CBBs. Unexpectedly, other players (SAK/PLK4, SPD2/CEP192 and CP110) emerged in a taxon-specific manner. We report that gene duplication plays an important role in the evolution of CBB components and show that, in the case of BLD10/CEP135, this is a source of tissue specificity in CBB and flagella biogenesis. Moreover, we observe extreme protein divergence amongst CBB components and show experimentally that there is loss of cross-species complementation among SAK/PLK4 family members, suggesting species-specific adaptations in CBB assembly. We propose that the UNIMOD theory explains the conservation of CBB architecture and that taxon- and tissue-specific molecular innovations, gained through emergence, duplication and divergence, play important roles in coordinating CBB biogenesis and function in different cellular contexts.