The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

Abstract

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.

Keywords: centromere disassembly; chromosome dynamics; heterochromatin decondensation; pollen tip growth; rDNA activation.

Fig. 1.

IGI is CDC48A and is required for active CenH3 removal from centromeres in the pollen vegetative cell and pollen tube formation. (A) Fluorescence microscopy of an A. thaliana IGI⁺ and an igi mutant mature pollen grain carrying pCenH3:CenH3:GFP and pLAT52:H2B:RFP. Approximately 1000 pollen vegetative cell nuclei from a +/igi heterozygous plant homozygous for pCenH3:CenH3:GFP and pLAT52:H2B:RFP were inspected, and ∼35% of them showed CenH3-GFP centromeric foci. SC, sperm cell; VN, vegetative cell nucleus. (Scale bar: 10 μm.) (B) Protein domain organization of A. thaliana CDC48A. Amino acid coordinates are indicated. The position of a substitution mutation in allele cdc48a-igi is marked with an asterisk. (C) Immunolocalization of CenH3 in WT, igi, cdc48a-1, and cdc48a-3 vegetative cell nuclei from mature pollen, using anti-CenH3/HTR12 antibodies. Nuclei were counterstained with DAPI (blue). (Scale bar: 1 μm.) Approximately 200 vegetative cell nuclei of pollen from +/igi and +/cdc48a plants were examined, and ∼20% of them showed CenH3 centromeric foci, whereas none of >1,000 WT vegetative cell nuclei showed this phenotype. (D) Pollen germination assays on solid media. Percentages of germinated (orange bar) and nongerminated (green bars) pollen grains from IGI⁺/igi heterozygous plants in the pCenH3:CenH3:GFP pLAT52:H2B:RFP background were examined for the igi phenotype showing centromeric CenH3-GFP dots in the RFP-positive vegetative cell nucleus under the fluorescence microscope. The pollen phenotype (IGI⁺ or igi) is indicated. Numbers on the bars indicate the number of pollen grains examined. (E) Fluorescence microscopy of a pollen grain at mononucleate microspore, dinucleate, and trinucleate mature stages from a transgenic plant homozygous for pCDC48A:CDC48A-BFP, pCenH3:CenH3:GFP, and pLAT52:H2B:RFP. (Scale bars: 10 μm.) All of ∼100 pollen grains at each stage showed an equivalent CDC48A expression pattern.

Fig. 2.

CenH3 is subjected to sumoylation, and CDC48A associates with sumoylated CenH3 in early-stage pollen. Coimmunoprecipitation of CDC48A-interacting proteins extracted from early-stage pollen of transgenic lines expressing CDC48A-Myc (A) and CDC48A-Myc and CenH3-GFP or CenH3-GFP without (−) CDC48A-Myc (B) using anti-Myc antibodies, followed by protein blot analyses with antibodies against CenH3, SUMO1, or Myc. Modified CenH3 band signals are marked by asterisks. S, sumoylation. (C) Immunoprecipitated proteins were fractionated by 4–20% gradient SDS/PAGE and stained with silver. Acrylamide gel slices s1–s7 were cut out and in-gel trypsin-digested peptides were extracted from each gel sample for MS analysis. SUMO1 was identified only in s1 (indicated with an asterisk), whose position corresponded to the modified CenH3 band in A. (D) Immunoprecipitation of CenH3-GFP from early-stage pollen extracts of the CDC48A-Myc CenH3-GFP line using anti-GFP antibodies, followed by protein blot analyses with antibodies against CenH3 and SUMO1. (E) Early-stage pollen proteins were immunoprecipitated from lines with or without (−) the pCDC48A:CDC48A:Myc transgene using anti-Myc antibodies, fractionated and silver-stained. Trypsin-digested peptides were extracted from gel slices s8–s13, which showed protein bands unique to the CDC48A-Myc line (Left), and from the WT control (Right) counterparts c8–c13, and were then analyzed by MS. The position of CDC48A-Myc bait protein is indicated with an asterisk. Proteins that were detected in both lines were excluded as false-positive findings. (F) CDC48A-associated cofactors and SUMO–proteasome pathway components were detected by MS. Spectra represent the number of all peptides that map to the accession indicated in the second column. The percentage coverage (% coverage) takes into account unique peptides that map only to the accession.

Fig. 3.

CDC48A and NPL4 cooperate to remove SUMO1-modified CenH3 from centromeres in the pollen vegetative cell. Dual-FISH/immunolocalization of 180-bp centromeric DNA repeats (180CEN) and SUMO1 in cdc48a-3 vegetative cell (A), WT vegetative cell (B), WT sperm (C), and npl4a npl4b vegetative cell (D) nuclei from mature pollen is shown. (Scale bars: 1 μm.) Approximately 300 pollen vegetative cell nuclei from +/cdc48a-3 and npl4a/npl4a +/npl4b plants were inspected, and ∼15% of them showed SUMO1 focal signals that colocalized with condensed 180CEN foci, whereas all of >500 WT vegetative cell nuclei showed dispersed SUMO1 signals.

Fig. 4.

CDC48A-directed CenH3 removal from centromeres is coupled to loss of H3K9me2 and centromeric heterochromatin decondensation. Dual immunolocalization of CenH3 and H3K9me2 (A, C, and E) and FISH analysis of 180CEN (B, D, and F) in WT, cdc48a-3, and npl4a npl4b vegetative cell nuclei from mature pollen is shown. (Scale bars: 1 μm.) Approximately 300 pollen vegetative cell nuclei from +/cdc48a-3 and npl4a/npl4a +/npl4b plants were examined, and ∼20% of them showed condensed CenH3, H3K9me2, and 180CEN foci, whereas in all of >1,000 WT vegetative cell nuclei, 180CEN repeats were dispersed and neither CenH3 nor H3K9me2 signal was detected. (G) Protein blot analyses of CDC48A-Myc–associated early-stage pollen proteins using antibodies to Myc and H3K9me2. See also Fig. 2B.

Fig. 5.

CDC48A-mediated centromeric heterochromatin decondensation coincides with activation of silent rRNA genes within the nucleolus. Dual-FISH analysis of 45S rDNA and 180CEN (A and C) and FISH/immunolocalization of 45S rDNA and fibrillarin (B and D) in WT leaf and mature pollen vegetative cell nuclei are shown. Note that leaf diploid nuclei typically show ten 180CEN and four rDNA focal signals. (Scale bars: 1 μm.) All of >1,000 pollen vegetative cell nuclei exhibited clustering of two rDNA loci that largely overlap the fibrillarin nucleolus marker. (E) Diagram illustrates the polymorphic regions of A. thaliana rDNA sequences in which insertions/deletions in the 3′ external transcribed region define the major rRNA gene variant types: VAR1, VAR2, VAR3, and VAR4. Arrows indicate positions of PCR primers used to detect these variants. Dashes indicate gaps in the alignment. (F) RT-PCR analysis of rRNA variants in WT leaf, total pollen, and FACS-purified vegetative cell nuclei (VN). RNA samples were treated with DNase before RT-PCR. Levels of expression were compared using equal amounts of input total RNA for RT-PCR. PCR without the reverse transcription step gave no PCR product. (G) Shotgun bisulfite sequencing analysis of DNA cytosine methylation in the CG context in the 5′ ETS region of 45S rDNA repeats from WT sperm and vegetative cell genomes. Positions of CG sites relative to the transcription start site (+1) are indicated beneath the graph. (H–K) FISH/immunolocalization of 45S rDNA, fibrillarin, 180CEN, and CenH3 in cdc48a-3 mutant vegetative cell nuclei from mature pollen. (Scale bars: 1 μm.) Approximately 300 pollen vegetative cell nuclei from +/cdc48a-3 plants were inspected in each experiment, and ∼20% of them showed two separate rDNA loci colocalizing with centromeres external to the nucleolus. Equivalent results were obtained with cdc48a-igi and cdc48a-1 alleles.