A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis

Abstract

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

Figure 1.

Arabidopsis NUC2 Is a Functional Protein Gene. (A) Photographs of Arabidopsis wild-type, nuc2-2 (GABI_178D01), nuc1-2 (SALK_002764), and nuc1 nuc2 plants. (B) Arabidopsis wild-type, nuc2-2, and nuc2-2 gNUC2 plants were grown on soil under a 16-h/8-h light/dark cycle. Late flowering is observed in nuc2-2 mutants compared with wild-type and nuc2-2 gNUC2 plants. (C) Top panel, RT-PCR using cDNA prepared from 15-d-old wild-type (lane 1), nuc2-2 (lane 2), and nuc2-2 gNUC2 (lane 3) seedlings to detect NUC2, NUC1, and 3′ ETS pre-rRNA transcripts, respectively. Amplification of eIF1α transcripts was performed to verify similar amounts of cDNA in each sample. Bottom panel, immunoblot reaction using α-At-NUC1. The membrane was stained with Ponceau S before hybridization to verify similar amounts of proteins. (D) RT-qPCR analysis to determine relative levels of FLC transcripts in wild-type, nuc2-2, and nuc2-2 gNUC2 plants. The drawing at bottom shows the Arabidopsis NUC2 gene from the ATG start codon to the TGA stop codon. Black boxes correspond to exons separated by introns. The positions of primers 5′nuc2/3′nuc2 and 5′nuc2q/3′nuc2q used in RT-PCR experiments are shown. A sequence upstream from ATG (nucleotide 1483), the first intron, and the exon from the NUC2 gene fused to GUS are represented. The positions of primers 5′nuc1/3′nuc1 and rRNA 5′3ets/3′3ets are shown in Figure 4 and Supplemental Figure 3. The T-DNA insertion in the nuc2-2 mutant plant GABI_178D01 is indicated. [See online article for color version of this figure.]

Figure 2.

NUC2 Gene Expression during Seedling Development and in Plant Organs. (A) RT-PCR to show NUC2 and NUC1 transcripts during seed germination (lanes 1 to 4), in shoots (lane 5), and in roots (lane 6). Absence of genomic contamination in the cDNA samples was verified by amplification of NUC1, NUC2, and eIF1α genomic DNA, which generates higher molecular size bands (lane 7). The number of cycles (24, 28, and 35) of each PCR is indicated. (B) Immunoblot experiment to determine the levels of NUC1 and NUC2 proteins during seed germination. α-Actin (ACT) was used to verify similar amounts of proteins. (C) RT-qPCR to determine the relative levels of NUC2 transcripts in shoots, roots, leaves, and flowers. (D) Analysis of NUC2 promoter activity in Arabidopsis plants transformed with the pAtNUC2:GUS construct. Top and middle rows, Arabidopsis seeds were sown on MS medium and maintained for 48 h at 4°C (day 0) and then transferred to room culture, at 22°C under continuous light conditions, for 4, 7, 10, and 15 d. Bottom row, GUS staining in leaf hydathode cells, roots, flowers, and buttress. The NUC2 gene fused to GUS is represented in Figure 1.

Figure 3.

NUC1 Participates in the Regulation of NUC2 Gene Expression. (A) Analysis of NUC2 promoter activity in nuc1 mutant plants transformed with the pNUC2:GUS construct. The bottom panels show shorter staining to better visualize GUS activity in leaves and roots. (B) Chromatin from wild-type plants was isolated and incubated either with protein A only (lanes 1, 4, and 7) or with antibodies against NUC1 (lanes 2, 5, and 8). Immunoprecipitated DNA was analyzed by PCR to detect NUC2 sequences from −466 to −210 (lanes 1 to 3), from −307 to −38 (lanes 4 to 6), and from −137 to +141 (lanes 7 to 9). The positions of primers used to amplify NUC2 sequences are indicated. The transcription initiation site is mapped by 5′ rapid amplification of cDNA ends at 87 nucleotides upstream from ATG. An aliquot of total chromatin sample before the immunoprecipitation reaction was used to control PCR amplifications (lanes 3, 6, and 9). Chromatin isolated from the nuc1 mutant was used to control the specificity of At-NUC1 antibodies in the ChIP reaction (nuc1-2 panels). (C) RT-PCR to show NUC2 and NUC1 transcripts in wild-type (lane 1) and met1-1 (lane 2) plants. The amount of cDNA was verified in each reaction by controlling eIF1α transcript levels. Absence of genomic contamination in the cDNA samples was verified by amplification of eIF1α genomic DNA, which generates higher molecular size bands (lane 3). (D) Immunoblot analysis to detect NUC1 and NUC2 proteins in nuc1 (lanes 1 and 2), nuc2 (lane 3), nuc2-2 gNUC2 (lane 4), met1-1 (lane 5), and wild-type (lane 6) plants. Similar amounts of protein in each membrane were verified by Ponceau S staining.

Figure 4.

Transcription and rDNA Organization in nuc2-2 Plants. (A) Primer extension was performed using total RNA extracted from wild-type (lane 1), nuc2-2 (lane 2), and nuc2-2 gNUC2 (lane 3) plants. Primer tis detects transcription from the TIS, and primer p detects pre-rRNA precursors cleaved at the primary cleavage site (P) (Sáez-Vasquez et al., 2004; Pontvianne et al., 2007). Primer extension using primer U3 that accurately maps the 5′ end of U3snoRNA was used to control similar amounts of total RNA in each reaction. (B) Immunoblot analysis to detect NUC1 and NUC2 protein in nuc1 (lanes 1 and 3) and wild-type (lane 2) chromatin extracts. The Coomassie blue gel staining reveals similar amounts of protein in each chromatin sample. (C) Immunolocalization of NUC2 in leaves from nuc1 plants. The NUC2 signal (green) colocalizes with chromatin situated in the periphery of the nucleolus and counterstained with DAPI (blue) (nuc1 panels). No NUC2 signal is detected in wild-type or nuc2 plants (used here to control the specificity of the antibodies). (D) FISH analysis with an rDNA probe containing sequences upstream and downstream from TIS (from −250 to +250) reveals the 45S rDNA loci (green) on nuclei from wild-type, nuc2-2, and nuc2-2 gNUC2 plants. Counterstaining with DAPI (blue) is shown (FISH and DAPI). The bar graphs at bottom show the percentage of rDNA loci associated with the nucleolus in wild-type, nuc2-2, and nuc2-2 gNUC2 plants. Bars = 5 μm. (E) PCR detection of rRNA gene variants in DNA of purified nuclei (N) or nucleoli (No) of wild-type (lanes 1 and 2) and nuc2-2 (lanes 3 and 4) plants. The positions of 5′3ets and 3′3ets primers used in PCR experiments are shown. The scheme at bottom represents the 45S rDNA unit containing the ETS (5′ ETS and 3′ ETS) and the structural rRNA sequences (18S, 5.8S, and 25S rRNA in gray boxes) separated by internal transcribed spacers (ITS1 and ITS2). Four repeat sequences located in the 3′ ETS are represented (R1 to R4). The TIS and the primary cleavage site (P) in the 5′ ETS are indicated. The positions of primers used to amplify or detect rRNA genes and/or pre-rRNA sequences are shown.

Figure 5.

The Relative Abundance of rDNA Variants Is Affected in Plants with a Disrupted NUC2. (A) Top, quantitative PCR analysis to amplify 18S, 25S, and ITS1 rDNA sequences from wild-type and mutant (nuc2-2, nuc2-2 gNUC2, nuc1-2, and fas2-4) plants. The bar graphs show relative amounts of 18S (black), 25S (dark gray), and ITS1 (gray) rDNA. Bottom, RT-PCR using cDNA prepared from 15-d-old wild-type (lane 1), nuc2-2 (lane 2), nuc1-2 (lane 3), and fas2-4 (lane 4) seedlings to detect 3′ ETS pre-rRNA transcripts. Amplification of eIF1α was performed to control similar amounts of cDNA in each sample. PCR on genomic DNA from wild-type plants detects the rDNA variants 1 to 4 (lane 5). (B) PCR amplifications of 3′ ETS sequences using genomic DNA from wild-type, nuc2-2, nuc2-2 gNUC2, nuc1-2, and fas2-4 plants. Relative amounts of each rDNA variant were determined using the LabChip GX system. The bar graphs show the percentage of rDNA VAR1 (black), VAR2 (gray), and VAR3 (dark gray).

Figure 6.

rDNA Is Hypermethylated in nuc2-2 Mutant Plants. (A) Bisulfite sequencing analysis. Top, the bar graphs show the percentage of methylated sites in the rRNA gene sequences (from −315 to +243) from wild-type, nuc2-2, and nuc2-2 gNUC2 plants in CG, CHG, and CHH contexts. Bottom, frequencies at which individual cytosines in a CG context are methylated between −315 and +243 relative to TIS. The scheme shows the positions of rDNA sequences upstream (promoter) and downstream (5′ ETS) of TIS. Most of the CG sites localize in the 5′ ETS region. (B) Genomic DNA from wild-type, nuc2-2, nuc2-2 gNUC2, and nrpe1 plants digested (lanes 5 to 8) or not (lanes 1 to 4) with McrBC. Amplifications of 3′ ETS, At LINE, At SN1, and 5S were performed to detect methylated or unmethylated DNA sequences in nuc2-2, nuc2-2 gNUC2, and control (wild-type and nrpe1) plants.

Figure 7.

Reintegration of Wild-Type rDNA Restores the Abundance of rDNA Variants and the Repression of VAR1 in nuc2-2. (A) Flowering time analysis of wild-type, nuc2-2, and backcrossed plants using pollen from nuc2-2 (nuc2 × wild type) or from wild-type (wild type × nuc2) plants. (B) RT-PCR using cDNA prepared from 15-d-old nuc2-2 (lane 1), wild-type (lane 2), +/nuc2 (lanes 3 and 4), and nuc2/+ (lanes 5 and 6) seedlings to detect 3′ ETS pre-rRNA transcripts. (C) The bar graphs show relative amounts of rDNA VAR1 (black), VAR2 (gray), and VAR3 (dark gray) (top) and 18S (black), 25S (gray), and ITS1 (dark gray) (bottom) rDNA sequences in the wild type, nuc2-2, nuc2/+ (BC4), and +/nuc2 (BC9). [See online article for color version of this figure.]

Figure 8.

NUC1 and NUC2 Binding and Remodeling of Nucleosomes. Centrally positioned nucleosomes were incubated with increasing (0.2, 0.4, 0.8, and 1.6 μL) amounts of RSC in the absence (lanes 2 to 5 and 12 to 15) or presence (lanes 7 to 10 and 17 to 20) of 300 ng of recombinant His-NUC1 or His-NUC2. Lanes 1 and 11 show nucleosomes alone, and lanes 6 and 16 show nucleosomes with 300 ng of His-NUC1 and His-NUC2, respectively. The migrations of centrally positioned nucleosomes, mobilized nucleosomes, and the ³²P radioactively end-labeled DNA are indicated on each EMSA gel.

Figure 9.

NUC2 Is Required for Chromatin Dynamics during Developmental Transitions. In this model, 45S rDNA VAR1 localizes in NOR2 (red) and VAR2 and VAR3 localize in NOR4 (green). Early in seed germination (2 d), both NOR2 and NOR4 are decondensed and transcriptionally active. NUC1 protein is associated with each NOR, as NUC1 binds to active genes (Pontvianne et al., 2010). Then, throughout germination, HDA6 participates in the chromatin silencing of rDNA VAR1 (Earley et al., 2010; Benoit et al., 2013). NOR2 becomes progressively silenced/condensed, and NUC1 dissociates. At 8 to 10 d, NUC2 transcripts and protein accumulate and bind “silenced” chromatin to participate in the heterochromatin establishment of NOR2. At the end of seed germination transition, NUC1 might play a role in repressing NUC2 gene expression at the transcriptional and/or posttranscriptional levels.