Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions

Abstract

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

Keywords: acrocentric chromosomes; nucleolar organizer regions (NORs); nucleolus; recombination; ribosomal DNA.

Figure 1.

NORs within monochromosomal hybrids retain functional potential. (A) Schematic diagram of the rDNA arrays on the p-arms of the five acrocentric human chromosomes. (PJ) Proximal junction, (DJ) distal junction. (B) Schematic diagram of the original 379-kb DJ contig showing the location of large inverted repeats and CER satellite. (C) List of monochromosomal hybrids, showing the human acrocentric chromosome they contain and the human cell line source of this chromosome. (D) A schematic representation of human SL1 and the mouse and human 5′ ETS RNA FISH probes. (E) RNA FISH experiments performed on hybrid cells transfected with expression plasmids encoding human TAF_I A–D.

Figure 2.

Sequencing of DJ regions from individual human acrocentric chromosomes. (A) Schematic representations of DJ contigs. Hybrid and chromosomal identities are shown on the left, together with GenBank accession numbers. (B) The average percentage identity of 100-kb blocks, among all seven DJ contigs is shown below the WAV17 (HSA21) contig. (C) Alignment of DJ contigs demonstrating that they can be clustered into three groups, based on the sequence of their distal ends. The percentage identity of the most distal sequences within group 1 and 2 members is shown schematically on the right. (D) A schematic representation of indel distribution on the left arm inverted repeat. Hybrid and chromosomal identities are shown on the left (see Supplemental Fig. S7B for sequence alignments at break points).

Figure 3.

DJ transcripts, arising from each acrocentric chromosome are essential for nucleolar function. (A) A schematic of the DJ showing location of left arm and right arm transcripts, disnor 187 and disnor 238, respectively. Below, results from RNA FISH performed on hTert-RPE1 cells with BAC clone CH507-145C22 (CU633906) to detect DJ transcripts, labeled in green, together with a human 5′ ETS probe in red. (B) Detection of disnor238 in monochromosomal somatic cell hybrid lines by RT-PCR with primers from exon 1 and 3. A9 and GM09142 (DJ-negative) cells served as negative controls and hTert-RPE1 as a positive control. (C) Twenty-nucleotide chimeric antisense oligonucleotides (ASO1 and 1M) comprise 5-nt 2-O-methoxyribonucleotide segments at both termini and a deoxynucleotide segment containing 10 central nucleotides. Inter-nucleotide linkages were phosphorothioate (*). hTert-RPE1 cells were transfected with ASO1 and 1M and analyzed 24 h later by immunofluorescent staining with antibodies against UBF and NOP52, combined with EU incorporation to monitor ongoing transcription. Effective depletion of DJ transcripts is demonstrated in Supplemental Figure S8. Representative cells, indicated as 1–3, are described in the main text. Quantitation of the experiment is shown below. A further two independent experiments provided essentially the same result (data not shown).

Figure 4.

Identification of “DJ-like” sequences in the chimpanzee, Pan troglodytes. (A) A schematic representation of the chimpanzee “DJ-like” contig, illustrating sequence composition and positions of BAC clones and cloned PCR products used in its construction. The percentage identity, 100-kb blocks, with human DJ contig from WAV17 (HSA21) is shown below. (B) DAPI (blue)-stained metaphase spreads prepared from B-cells of two individual chimpanzees, Vanessa (female) and Zeef (male), probed with a mixture of “DJ-like” BAC clones CH251-114B1 and CH251-351B7 (green) and a human rDNA probe (red). Insets show enlarged acrocentric chromosomes. (C) Individual acrocentric chromosomes from Vanessa (female) and Zeef (male) metaphase spreads probed with “DJ-like” chimpanzee BAC clones (red), human PJ BAC clone bP-2154M18 (CR381535) (green), and rDNA (far red, pseudocolored here in blue). Chromosomes were DAPI-stained (pseudocolored in gray). Full metaphase spreads are presented in Supplemental Figure S11B. (D) A schematic illustrating the inverted orientation of rDNA and “DJ-like” sequences in chimpanzees.

Figure 5.

Far-distal regions exhibit variation between acrocentric chromosomes. (A) A schematic illustrating the proposed location of sequences present in BAC clone AL591856 relative to the DJ contig from A9-15 (HSA15). (B) A normal human female (F5) metaphase spread probed with BAC clone AL591856 (green) and alpha satellite probes recognizing HSA13/21 (red) and HSA14/22 (far red, pseudocolored here in blue). Chromosomes were DAPI-stained (pseudocolored in gray). Enlarged versions of all 10 acrocentric p-arms are shown on the right. (C) Human acrocentric chromosomes present in hybrid lines show varying composition of AL591856-like sequences in far-distal regions. The portions of DJ contigs and separate contigs that have high sequence identity with far-distal BAC clone AL591856 are shown below a schematic representation of this BAC. These fall into the same three group classifications described in Figure 3C. Coordinates and perent identities are indicated. The red asterisk beside group 1 member A9-22 indicates the presence of a second contig with lower, but still significant, homology (∼87%) with AL591856 (see Supplemental Fig. S13).

Figure 6.

Far-distal regions exhibit variation among human individuals. Metaphase spreads from six human individuals; M (male) and F (female) and hTert-RPE1 cells were probed as above. Shown here are the p-arms of 10 acrocentric chromosomes present in a single representative spread from each individual. Full spreads are presented in Supplemental Figure S15. The relative signals from the AL591856 probe in each spread were classified according to an arbitrary four-point scale and shown in tabular format below. Results from F5 above were also included.