Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

Abstract

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain.

Keywords: antisense transcription; boundary elements; chromatin; gene silencing; nuclear organization.

Fig. 1.

Inhibition of gene expression and recombination by an rDNA repeat. (A) Mating-type region. The mat2-P and mat3-M cassettes are in a ∼20-kb heterochromatic region flanked by the IR-L and IR-R boundary elements. They are silenced and used for gene conversions of mat1 leading to mating-type switching. The IR-R boundary was replaced with an rDNA repeat in rDNA-R strains. rDNA repeats consist of a transcribed region encoding the 18S, 5.8S, and 28S rRNA, and an untranscribed spacer containing an origin of replication (autonomously-replicating sequence, ars) and binding sites for the Reb1 and Sap1 proteins that block replication fork progression. (B) Tenfold serial dilutions of (EcoRV)::ade6⁺ cells with a wild-type boundary (IR-R⁺, MAM56), a deletion of the boundary (IR-RΔ, MAM46), or rDNA-R (MAM36) were spotted on selective media. Cells lacking (EcoRV)::ade6⁺ are shown for comparison [IR-R⁺(WT), MAM26]. Cells expressing ade6⁺ grow on AA-ade and form white colonies on yeast extract (YE) plates. (C) (EcoRV)::ade6⁺ expression measured by quantitative RT-PCR relative to the expression of ade6⁺ at its normal chromosomal location (IR-R⁺, PM7; IR-RΔ, PM3; rDNA-R, PM2). (D) Mating-type switching was assayed by iodine staining of colonies grown on minimum sporulation agar (MSA). Dark staining is indicative of efficient switching because S. pombe spores, but not vegetative cells, are stained brown by iodine vapors. Light staining of rDNA-R colonies reveals a mating-type switching defect. Strains as in B. (E) Southern blot of genomic DNA digested with HindIII and probed with a mat1 probe (10.5-kb HindIII fragment). The fragile site at mat1 gives rise to a double strand break (DSB). (Left) Strains as in B. (Right) swi1Δ strains (IR-R⁺, PM18; rDNA-R, PM20). (F) Quantification of mat1 content by PCR. From left to right: IR-R⁺, MAM56; IR-RΔ, MAM46; rDNA-R, MAM36; and IR-R⁺(WT), MAM26.

Fig. 2.

Relocalization of the mating-type region to the nucleolus in rDNA-R cells. (A) Dimensions of S. pombe cells. The distance d between the mating-type region (yellow) and the center of the nucleolus (blue) was measured in this experiment. (B) The S. pombe cell cycle. G2 cells (highlighted in blue) were used for distance measurements. (C−F) Fluorescence images (Left) and distance measurements (Right) for strains with the indicated genotypes. The distance d was determined in 3D for the reported number of cells. Mean values (d̅) and SDs are indicated. The IR-R⁺(WT) strain shown in C has a wild-type mating-type region; the strains shown in D−F have an (EcoRV)::ade6⁺ insertion. A Student’s t test showed that the mating-type region is significantly closer to the nucleolus in the rDNA-R mutant than in IR-R⁺ or IR-RΔ strains; P values comparing each distribution to C are indicated in D−F.

Fig. 3.

Reb1 mediates the relocalization of the rDNA-R mating-type region to the nucleolus and its silencing. (A) Distribution of the mating-type region to nucleolus distance d for (EcoRV)::ade6⁺ cells propagated in the presence or absence of adenine as indicated. The red dotted lines correspond to double Gaussian fits (mean values d̅1 and d̅2) indicating the presence of two subpopulations of cells differentially associated with the nucleolus. (B) Deletion of reb1 derepresses (EcoRV)::ade6⁺ in rDNA-R but not in IR-R⁺ cells. Spot tests as in Fig. 1B. From top to bottom: PM8, PM107, PM53, PM59, PM67, and PM51. (C) ade6⁺ expression measured by quantitative RT-PCR as in Fig. 1C. WT ade6⁺, 968; WT ade6⁺ reb1Δ, PG3772; IR-R⁺, PM8; IR-R⁺ reb1Δ, PM107 and PM108; rDNA-R, PM59; rDNA-R reb1Δ, PM67-69. (D) Relative localization of Reb1 (tagged with mCherry and expressed from endogenous locus; shown in red) and mating-type region (GFP; shown in green) in IR-R⁺ (PM131), IR-RΔ (PM132), and rDNA-R (PM127) cells. Reb1 and the mating-type region colocalize in a fraction of the rDNA-R cell population.

Fig. 4.

Antisense transcription in the mating-type region of rDNA-R cells. (A) The positions of seven primer pairs used in B and C are indicated above the mating-type region. (B) Antisense transcription (i.e., polarity opposite to ade6⁺) was detected by real-time RT-PCR at the seven positions assayed in rDNA-R (PM59) but not in IR-R⁺ (PM8) cells. Transcript levels were estimated relative to act1⁺. (C) Sense and antisense (EcoRV)::ade6⁺ transcripts were measured with primer pair 1 in independent biological isolates. Three independent isolates were examined for rDNA-R reb1⁺, five for rDNA-R reb1Δ cells propagated in the presence of adenine, and three for rDNA-R reb1Δ cells propagated in the absence of adenine. Each RT-PCR was set up in triplicate. Transcript levels were normalized to ade6⁺ expressed from its normal chromosomal location. No anticorrelation between sense and antisense transcription was observed, indicating that antisense transcription is not, or not solely, responsible for changes in sense transcript abundance.

Fig. 5.

Relocalization and repression of the mating-type region by Reb1-binding sites. (A−E) Distribution of the mating-type region to nucleolus distance d for strains with the indicated genotypes. The mating-type region was associated with the nucleolus in a fraction of the 11xRBS (A) and 11xRBS clr4Δ (C) populations, leading to shorter mean distances than for 11xRBS reb1Δ cells (B) where the association was lost. The nucleolar association of rDNA-R remained in clr4Δ (E). Student t tests showed that the distributions in A and B were statistically different from each other (P < 1 × 10⁻⁴), as were the distributions in B and C (P < 1 × 10⁻⁴), whereas A and C were not (P = 0.05). The strains were, from A to E, TP360, TP361, TP362, PM16, and PM14. (F) Schema depicting the replacement of the IR-R⁺ boundary with 11 Reb1-binding sites (11xRBS). (G) The 11xRBS represses (EcoRV)::ade6⁺ in a Reb1- and Clr4-dependent manner. Spot tests as in Fig. 1B with, from top to bottom, PM8, TP360, TP361, TP362, and PM3. (H) (EcoRV)::ade6⁺ sense and antisense transcripts measured by quantitative RT-PCR normalized to euchromatic ade6⁺ sense expression as in Fig. 1C. The 11xRBS, but not rDNA-R, required the methyltransferase Clr4 for its silencing effects, and an antisense (EcoRV)::ade6⁺ transcript was abundant in rDNA-R cells, but not in 11xRBS cells. The means of several biological isolates are presented for each genotype. Quantification for each individual isolate is presented in Fig. S1. Strains, from left to right: PM8, PM107-108, PM31, TP360, TP361, TP362, PM59, PM67-69, and PM27. (I) Example of fluorescence images used for quantification in A and B.

Fig. 6.

Model. Insertion of an rDNA repeat in the mating-type region (rDNA-R) in the place of a boundary element (IR-R⁺) leads to a relocalization of the region away from its natural location at the spindle-pole body to the perinucleolar compartment. Perinucleolar association is stabilized by the dimerization of the myb-domain protein Reb1. This brings the mating-type region under the influence of macromolecules predominantly abundant or active in the nucleolus resulting in gene silencing.