Artificial generation of centromeres and kinetochores to understand their structure and function

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Abstract

The centromere is an essential genomic region that provides the surface to form the kinetochore, which binds to the spindle microtubes to mediate chromosome segregation during mitosis and meiosis. Centromeres of most organisms possess highly repetitive sequences, making it difficult to study these loci. However, an unusual centromere called a “neocentromere,” which does not contain repetitive sequences, was discovered in a patient and can be generated experimentally. Recent advances in genome biology techniques allow us to analyze centromeric chromatin using neocentromeres. In addition to neocentromeres, artificial kinetochores have been generated on non-centromeric loci, using protein tethering systems. These are powerful tools to understand the mechanism of the centromere specification and kinetochore assembly. In this review, we introduce recent studies utilizing the neocentromeres and artificial kinetochores and discuss current problems in centromere biology.

Introduction

In all organisms, cells undergo cell-division cycles to grow and to transmit chromosomes comprising the genetic information to the next generation. Chromosomes are segregated into daughter cells during the cell-division. The centromere plays an important role in chromosome segregation [[1], [2], [3]], and is a genomic region, at which the kinetochore is formed for interaction with spindle microtubules. Centromere position is usually maintained stably through multiple rounds of cell-division. If the centromere is shifted to another genome locus or is formed at multiple positions on a chromosome (multi-centromere), aberrant chromosome segregation occurs during mitosis and meiosis, which leads to cell death. However, a new centromere sometimes forms on a chromosome at a non-centromere locus, while the endogenous centromere is inactivated. The de novo formed centromere is called a “neocentromere” on which a normal kinetochore structure is formed [4,5]. The case of a centromere moving from one place to another on a chromosome was first reported in 1993 in a patient with mild developmental delay [5].

As the neocentromere is newly formed on a non-centromeric region, typical centromere repetitive sequences such as human alpha-satellite repeats, are not detected. Therefore, the discovery of neocentromeres supports the hypothesis that centromeres are specified by sequence-independent epigenetic mechanisms in most organisms [[5], [6], [7]]. Importantly, neocentromeres can be generated experimentally in various organisms [[8], [9], [10], [11]]. Currently, several epigenetic markers have been identified in and around the centromere, such as histone modifications, histone variants, and DNA modifications [4,[12], [13], [14]]. Among them, the centromere-specific histone H3 variant centromere protein A (CENP-A) has been extensively studied [[15], [16], [17]]. Many groups have been studied the mechanisms regarding CENP-A incorporation into centromeres [[18], [19], [20], [21], [22], [23], [24], [25]], and neocentromeres have been used in these studies.

Genetic, cell biology, biochemistry, and structural biology approaches have been used to study centromere specification and kinetochore assembly. Among them, neocentromeres have been a useful model system to study centromere epigenetics. Currently, the questions surrounding centromere epigenetics are shifted toward the chromatin conformation at the core centromere region. Certain significant and critical questions remain to answered:

  • 1.

    What is the trigger (DNA sequence or chromatin conformation) for the de novo formation of a centromere?

  • 2.

    What is the additional mark to form or specify a centromere, in addition to CENP-A?

  • 3.

    How are centromere position and size regulated?

  • 4.

    What is the role of centromeric proteins in establishing a functional kinetochore?

Recent approaches using next-generation sequencing (NGS) techniques have been utilized to examine chromatin structure including epigenetic features and 3D contact-map information at the whole genome to understand specific biological functions in the genome [[26], [27], [28]]. However, centromere regions are usually omitted from genome analyses, because normal centromeres contain long and highly repetitive sequences in most organisms. Although recent NGS analysis offers better models of human alpha-satellite sequences at centromeres [29], it is still hard to apply the NGS-based approach to repetitive centromeres. In this review, we introduce de novo centromere formation on non-repetitive DNA sequences, including natural and experimentally generated neocentromeres, and summarize the studies regarding the above questions using non-repetitive neocentromeres. We also introduce artificial minimal kinetochores to understand the mechanisms of the kinetochore assembly.

Section snippets

Neocentromere formation in natural environments

The first report of a human neocentromere was published in 1993 [5]. Patients with a certain kind of mental retardation carry a chromosome called “mar del (10) chromosome”, which is derived from a normal chromosome 10 and has an atypical centromere lacking a centromere-specific alpha-satellite sequence. Despite lacking an alpha-satellite sequence, the mar del (10) chromosome was segregated normally through multiple cell-divisions. Later, detailed sequence analyses revealed that the centromere

DNA features at native centromeres

The centromere provides a microtubule attachment site for chromosome segregation during mitosis. The microtubule-binding protein complex on the centromere, called the “kinetochore”, consists of numerous proteins including CCAN (constituting centromere-associating network) proteins and KMN (Knl1 complex-Mis12 complex-Ndc80 complex) network proteins [1,15,52]. Although studies on kinetochore proteins have progressed, the centromeric chromatin structure is largely unknown. As mentioned above, new

The contribution of heterochromatin to centromere formation and function

The core centromere contains CENP-A nucleosomes with interspersed H3-containing nucleosomes and pericentromeric heterochromatin around the core centromere [[85], [86], [87], [88], [89]] (Fig. 4A). Histone modification profiles were examined at core centromeres and pericentromeres, based on chromatin fiber-based immunostaining analyses. At the core centromere, H3K4-me2 was found to be accumulated, but there were fewer heterochromatin marks such as H3K9-me2, -me3, and H3K4-me3, while they were

Regulation of centromere size

The size of a centromere and its position should be stably transmitted to the next generation of cells during mitosis and meiosis in most organisms. An unstable centromere size might lead to imbalanced microtubule attachment and biased chromosome segregation, as observed in meiotic-drive or inverted-meiosis phenomena [43,122,123]. Therefore, the size of a centromere must be primarily regulated by CENP-A levels in addition to various layers of regulation for centromere size, depending on cell

Minimum artificial kinetochore

The kinetochore structure consists of two major protein networks including the CCAN and KMN network. The CCAN establishes a chromatin base for the kinetochore, and the microtubule-binding KMN network is recruited to CCAN during mitosis [1,15,52] (Fig. 6A). Among CCAN proteins, CENP-C and CENP-T play an important role in KMN recruitment, because CENP-C and CENP-T directly bind to the Mis12 and Ndc80 complexes of KMN, respectively [1,15,52] (Fig. 6A).

The tethering of kinetochore components to a

Concluding remarks

The centromere and kinetochore are essential for chromosome segregation; therefore, their characterization is important. However, the complicated features of natural centromeres and kinetochores make them difficult to characterize. Instead, the use of artificial neocentromeres and kinetochores, which can be experimentally generated, has increased our understanding of centromeres and kinetochores. Neocentromeres and centromeres can be moved, suggesting that centromere formation might be

CRediT authorship contribution statement

Tetsuya Hori: Writing - review & editing. Tatsuo Fukagawa: Writing - review & editing.

Acknowledgments

The authors are very grateful to all members of the Fukagawa Lab for useful discussions. This work was supported by JSPS KAKENHI Grant Number 25221106, 17H06167, 16H06279 and 15H05972 to TF, JSPS KAKENHI Grant Number 17K07501 to TH.

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