Allelic inactivation of rDNA loci

Abstract

Human cells contain several hundred ribosomal genes (rDNA) that are clustered into nucleolar organizer regions (NORs) on the short arms of five different acrocentric chromosomes. Only approximately 50% of the gene copies are actually expressed in somatic cells. Here, we used a new cytological technique to demonstrate that rDNA is regulated allelically in a regional manner, with one parental copy of each NOR being repressed in any individual cell. This process is similar to that of X-chromosome inactivation in females. Early in development, one copy of each NOR becomes late-replicating, thus probably marking it for inactivation and subsequent targeted de novo methylation at rDNA promoter regions. Once established, this multichromosomal allelic pattern is then maintained clonally in somatic cells. This pathway may serve as an epigenetic mechanism for controlling the number of available rDNA copies during development.

Figure 1.

ReTiSH. (A) Scheme of ReTiSH method showing how BrdU-labeled regions are converted to ssDNA so that they can be identified by hybridization with specific probes. MEFs were labeled with BrdU for 7 or 12 h, arrested in metaphase, and subjected to ReTiSH using BAC probes for the late-replicating gene Adyc8 (RP23-352F10) (B) or the early-replicating gene A2M (RP23-191C6) (C). (D) Human lymphoblasts were labeled with BrdU for 7 or 12 h, arrested in metaphase, and subjected to ReTiSH using a BAC probe (RP11-55G7) for the asynchronously replicating olfactory receptor (family 4) gene cluster on chromosome 14 (OR4). Arrows indicate relevant gene signals.

Figure 2.

Replication timing of rDNA. (A) Human lymphoblasts or foreskin fibroblasts were labeled with BrdU for various times, arrested in metaphase, and subjected to ReTiSH using a probe for rDNA. The number of rDNA signals in each metaphase spread (n = 461) was counted, and standard deviation was calculated for each data point (see error bars). (B) Examples at 7 and 12 h are shown.

Figure 3.

Asynchronous replication timing is allelic. Human lymphoblasts were labeled with BrdU for 7 h, arrested in metaphase, and subjected to ReTiSH using probes for rDNA (green) and the subcentromeric regions on chromosome 14, 15, 21, or 22 (red). (A) The percentage of metaphase spreads (n = 431) showing rDNA hybridization on a single copy of the labeled chromosome was recorded. (B) Examples of metaphase spreads. Early-replicating alleles were observed using a pulse-chase BrdU-labeling protocol (Materials and Methods). Arrows indicate locations of polymorphic subcentromeric regions. (C) Pools and single-cell subclones of human lymphoblasts with polymorphic centromeric or subcentromeric regions on chromosomes 14, 15, and 22 were analyzed by ReTiSH using probes for ribosomal and centromeric DNA. The number of metaphases (n = 430) with maternal late replication of the rDNA is recorded. The probability of having a single copy of any particular chromosome labeled by ReTiSH in middle S is five out of nine (0.555). The P-value was calculated by the hypergeometric method, (0.555)³⁷⁰ × (0.445)⁶⁰ × 430!/(370!60!) = 0.5 × 10⁻⁴².

Figure 4.

Visualization of histone modification on metaphase chromosomes. (A) Metaphase spreads from YC lymphoblasts were visualized by immunohistochemistry with fluorescent antibodies (red) either to H3Ac (left) or H3K4me (right). The magnified area presents the histochemistry signal underlying two rDNA loci, one positive and one negative for H3K4me. The number of rDNA sites colocalized (yellow) with H4Ac, H3Ac, or H3K4me were counted (n = 78) and presented in graphic form as the percent of the rDNA loci labeled with modified histones. Arrows indicate colocalization (yellow) of rDNA and H3Ac or H3K4me. (B) Metaphase spreads from human YC lymphoblast clones (2 or 127) were used for immunohistochemistry (red) with anti-H4Ac or anti-H3K4me and then hybridized with a probe for the centromere on chromosome 15. In this experiment, the centromeric region is small on the maternal allele (open arrow) and large on the paternal allele (closed arrow). In each nucleus, the H3K4me-positive allele (red) is labeled with a white arrow. These results, including the number of nuclei examined, are summarized in the table. The maternal allele is early-replicating in clone 127 and late-replicating in clone 2.

Figure 5.

Chromosomal coordination of rDNA asynchronous replication. (A) FISH analysis of human lymphoblast clone 2 BrdU-labeled (gray) nuclei using one probe (red) for the chromosome 14 subcentromeric region and another for an olfactory receptor cluster (green) located very close on this same chromosome (14q11.2). The early-replicating (double) olfactory receptor gene was associated with the larger paternally derived subcentromeric region on 17 out of 20 asynchronous nuclei, and this is the same parental chromosome that has an early-replicating NOR in these same cells (Fig. 3C). ReTiSH analysis of human lymphoblast clones 127 (B) and 2 (C) labeled with BrdU for 7 h and arrested in metaphase using a rDNA probe (green), a chromosome 15 olfactory receptor (15q26) probe (red), and a centromeric probe (red). In B, the late-replicating olfactory receptor locus and late-replicating rDNA locus are both associated with the smaller maternal centromeric region (19 out of 21), while in C, both late-replicating loci are associated with the larger paternal subcentromeric region (13 out of 19), and this is consistent with the data in Figure 3C. The parental alleles are also shown in the accompanying magnification. (D) ReTiSH analysis of human lymphoblast cells labeled with BrdU for 7 h and arrested in metaphase using a rDNA probe (green) and a chromosome 21 olfactory receptor probe (red). The labeled late-replicating olfactory receptor locus is almost always (22out of 23) associated with the same parental chromosome carrying a late-replicating rDNA locus (see magnification). Arrows indicate rDNA loci.

Figure 6.

Regulation of rDNA loci during embryogenesis. (A) MEFs, mouse ES cells, and mouse blastocyst cells were labeled with BrdU for various times; arrested in metaphase; and subjected to ReTiSH using a probe for rDNA. The number of rDNA signals in each metaphase spread (n = 193) was counted, and the standard deviation was calculated for each data point (error bars). The data were adjusted to account for the shorter G2 (2 h) in blastocysts, as determined by the time at which 50% of metaphases are labeled with BrdU. (B) Diagram showing the stereotypic rDNA repeat indicating the transcription start site and the internal (ITS) and external (ETS) spacer regions. Methylation of the 5′ rDNA region was determined by bisulfite analysis on individual molecules (Supplemental Fig. 1), and the results are recorded as a function of position. The numbers of unmethylated (tan) and fully methylated (brown) molecules are shown. (C) Levels (±SD) of rRNA from mouse blastocysts and adult brain or spleen were measured by quantitative RT–PCR and normalized against a set of three housekeeping genes. (D) Metaphase spreads from mouse cells were used for immunohistochemistry with anti-H3Ac or anti-H3K4me and then hybridized. Results show the percent of rDNA loci in each nucleus (n = 127) that are labeled with H3Ac or H3K4me.