The centromere is the nucleoproteic chromosomal structure necessary for accurate chromosome segregation during cell division. One of the earliest centromeric proteins to be discovered was CENP-B, the ...only one capable of recognizing a specific centromeric DNA binding motif. The phylogenetic history of this protein and of its DNA binding site shows independent events of function acquisition across different species and raises questions on the evolutionary dynamics of CENP-B, including what may be the selective advantage provided by its role at the centromere. Recent results have provided insight into potential functions of CENP-B in chromosome dynamics, however, its function is still object of debate. The recurrent appearance of CENP-B centromeric activity along phylogenesis, together with its dispensability, represent strictly intertwined facets of this controversy. This chapter focuses on the evolution, function and homeostasis of CENP-B and its importance in centromere biology.
The centromere performs a universally conserved function, to accurately partition genetic information upon cell division. Yet, centromeres are among the most rapidly evolving regions of the genome ...and are bound by a varying assortment of centromere-binding factors that are themselves highly divergent at the protein-sequence level. A common thread in most species is the dependence on the centromere-specific histone variant CENP-A for the specification of the centromere site. However, CENP-A is not universally required in all species or cell types, making the identification of a general mechanism for centromere specification challenging. In this review, we examine our current understanding of the mechanisms of centromere specification in CENP-A-dependent and independent systems, focusing primarily on recent work.
Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade ...AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with first-order kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.
Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, ...CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.
Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements known as chromothripsis, but the underlying mechanisms remain unresolved. Here we developed an ...inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimaera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated following centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harbouring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJ-mediated reassembly in the subsequent interphase.
Mitotic errors lead to aneuploidy, a condition of karyotype imbalance, frequently found in cancer cells. Alterations in chromosome copy number induce a wide variety of cellular stresses, including ...genome instability. Here, we show that cancer cells might exploit aneuploidy-induced genome instability and the resulting gene copy-number changes to survive under conditions of selective pressure, such as chemotherapy. Resistance to chemotherapeutic drugs was dictated by the acquisition of recurrent karyotypes, indicating that gene dosage might play a role in driving chemoresistance. Thus, our study establishes a causal link between aneuploidy-driven changes in gene copy number and chemoresistance and might explain why some chemotherapies fail to succeed.
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•Genome instability driven by aneuploidy can facilitate chemoresistance•Chemoresistant cells are characterized by recurrent karyotypes•Resistance to chemotherapy is dictated by changes in gene copy number•Chemoresistance is achieved through altered expression of specific proteins
Ippolito et al. show that induction of chromosome mis-segregation leads to increased karyotypic heterogeneity that could be exploited by cancer cells to survive during chemotherapy. Chemoresistant cells harbor recurrent karyotypes, which impose gene copy-number changes and consequent altered expression of specific proteins crucial for chemoresistance.
In order to maintain cell and organism homeostasis, the genetic material has to be faithfully and equally inherited through cell divisions while preserving its integrity. Centromeres play an ...essential task in this process; they are special sites on chromosomes where kinetochores form on repetitive DNA sequences to enable accurate chromosome segregation. Recent evidence suggests that centromeric DNA sequences, and epigenetic regulation of centromeres, have important roles in centromere physiology. In particular, DNA methylation is abundant at the centromere, and aberrant DNA methylation, observed in certain tumors, has been correlated to aneuploidy and genomic instability. In this review, we evaluate past and current insights on the relationship between centromere function and the DNA methylation pattern of its underlying sequences.
Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin ...assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A’s amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes with CENP-B-containing centromeres. These data demonstrate a DNA sequence-specific enhancement by CENP-B of the fidelity of epigenetically defined human centromere function.
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•CENP-B binding to alphoid DNA repeats stabilizes CENP-C and kinetochore nucleation•Centromere function is enhanced by mutual dependencies of CENP-A, CENP-B, and CENP-C•The CENP-B free Y and neocentromere chromosomes mis-segregate at elevated frequencies•CENP-B binding to alphoid DNA enhances fidelity of epigenetically defined centromeres
Fachinetti et al. uncover the functional importance of CENP-B, the only human centromere protein known to bind in a DNA sequence-dependent manner. The authors show that by directly enhancing CENP-C recruitment and CENP-C-dependent nucleation of kinetochore assembly, CENP-B increases the fidelity of epigenetically defined human centromere function.
Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor ...cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.
Centromeres are unique chromosomal domains that control chromosome segregation, and are epigenetically specified by the presence of the CENP-A containing nucleosomes. CENP-A governs centromere ...function by recruiting the constitutive centromere associated network (CCAN) complex. The features of the CENP-A nucleosome necessary to distinguish centromeric chromatin from general chromatin are not completely understood. Here we show that CENP-A undergoes α-amino trimethylation by the enzyme NRMT in vivo. We show that α-amino trimethylation of the CENP-A tail contributes to cell survival. Loss of α-amino trimethylation causes a reduction in the CENP-T and CENP-I CCAN components at the centromere and leads to lagging chromosomes and spindle pole defects. The function of p53 alters the response of cells to defects associated with decreased CENP-A methylation. Altogether we show an important functional role for α-amino trimethylation of the CENP-A nucleosome in maintaining centromere function and faithful chromosomes segregation.
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