The past, present, and future of human centromere genomics

Abstract

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function.

Keywords: CENP; alpha satellite; chromosome truncation; dicentric; heterochromatin; higher order repeat; human artificial chromosome; tet operon; transcription.

Figure 1

The genomic organization of human centromeres. The primary sequence at human centromeres is alpha satellite DNA that is based on 171 bp monomers (colored arrows) organized in a tandem head-to tail fashion. The monomeric sequences differ by as much as 40%. A set number of monomers give rise to a higher order repeat (colored bars with black arrowhead) and confer chromosome-specificity. Higher order repeats are themselves reiterated hundreds to thousands of times, so that the alpha satellite arrays are highly homogenous and span several hundred kilobases to several megabases. Unordered monomeric alpha satellite DNA flanks the higher order arrays, becoming progressively more divergent farther away from centromeric core.

Figure 2

Heterogeneity of alpha satellite DNA. The alpha satellite DNA at centromeres exhibits several types of polymorphism. (A) Total array size, defined by the number of higher order repeats (HOR; gray arrows), varies between homologues and among individuals; (B) The same alpha satellite array from a given chromosome type can contain HORs of different sizes. In addition, the number of each HOR variant can vary. For example, an alpha satellite array can contain a mixture of 10-mers and 6-mers, with a greater number of 10-mers. Another array from the same chromosome in a different individual might have an equal number of 10-mers and 6-mers or, alternatively, more 6-mers than 10-mers; (C) Alpha satellite DNA can also vary at the level of monomer (black arrowheads) type and arrangement. Some monomers (gray arrowheads) contain a specific sequence element called the CENP-B box. Others monomers can contain identical nucleotide changes or SNPs (yellow arrowheads) within the same array. Multiple SNPs (hot pink, orange, gray, yellow arrowheads) can be present in the same HOR or distributed across an alpha satellite array. Each type of variation (array size, HOR size, SNPs) is not mutually exclusive and all contribute to the heterogeneity of alpha satellite DNA in the human population.

Figure 3

Minichromosome-based assays defining alpha satellite as the functional human centromere. (A) In the late 1990s, human artificial chromosome (HAC) assays (bottom up approach) were developed to test the ability of alpha satellite DNA to form de novo centromeres. Synthetic or clone arrays of alpha satellite DNA, such as D17Z1 from human chromosome 17 (green), were cloned into bacterial or plasmid (P1) artificial chromosome (BAC/PAC) vectors containing selectable marker genes (SM). The chromosome assembly constructs were introduced by transfection into human cells. In approximately half of the cells, an autonomous de novo chromosome (arrowhead) was produced, consisting of the same alpha satellite DNA (D17Z1, green, as shown) as the parental chromosome (arrow). Inset shows DAPI (DNA) staining of HAC. In the other proportion of transfected clones, the alpha satellite assembly BAC/PAC vector does not make a HAC, but integrates once or multiple times (as shown) into one or more chromosomes. In these instances, the alpha satellite DNA does not recruit any, or all, centromere proteins and is not a functional centromere. Inset shows DAPI (DNA) stained chromosome that contains multiple insertions of D17Z1. (B) In a complementary top-down approach, existing chromosomes (X and Y) were systematically deleted using plasmid constructs containing mammalian telomeric sequence (yellow arrowheads). These experiments yielded partially deleted chromosomes with integrated telomeres (red-orange-yellow rectangles) that were progressively deleted. Mitotic chromosome segregation of these minichromosomes was used as a measure of chromosome stability. Based on the molecular composition of the stable minichromosomes that were recovered, alpha satellite DNA (pink oval) was defined as the minimal sequence required for centromere function.

Figure 4

The detailed genomic organization of the human X centromere. The first contiguous genomic map of a human centromere (CEN) on the X chromosome was completed in 2001 and showed that the higher order array (large light gray arrays containing black monomer arrowheads) is flanked by unordered, monomeric alpha satellite DNA (multi-colored arrows). The regions between monomeric alpha satellite and the chromosome short (Xp) and long (Xq) arms contain other types of satellite DNA, such as gamma satellite and HSAT4. LINEs (red lollipops) and SINEs (purple lollipops) punctuate the repetitive DNA between the centromere and chromosome arms. The Xq pericentromere contains monomeric alpha satellite and a LINE element at the pericentromere-arm junction. Some of the monomers within the unordered Xq satellite contain CENP-B boxes (black asterisks). The functional significance of these monomers remains unclear.

Figure 5

Timeline of major discoveries in human centromere genomics. Since the discovery of alpha satellite DNA in 1979, the understanding of the sequence, organization, and functional aspects of this sequence flourished during the Human Genome Project era. Recent years have shown the use of human artificial chromosomes (HACs) and the creation of the first database of alpha satellite sequences linked to their functional capacity.