ESET methylates UBF at K232/254 and regulates nucleolar heterochromatin plasticity and rDNA transcription

Abstract

The remodeling of chromatin in the nucleolus is important for the control of ribosomal DNA (rDNA) transcription and ribosome biogenesis. Herein, we found that upstream binding factor (UBF) interacts with ESET, a histone H3K9 methyltransferase and is trimethylated at Lys (K) 232/254 by ESET. UBF trimethylation leads to nucleolar chromatin condensation and decreased rDNA transcriptional activity. UBF mutations at K232/254A and K232/254R restored rDNA transcriptional activity in response to ESET. Both ESET-ΔSET mutant and knockdown of ESET by short hairpin RNA reduced trimethylation of UBF and resulted in the restoration of rDNA transcription. Atomic force microscopy confirmed that UBF trimethylated by ESET modulates the plasticity of nucleolar chromatin. We further demonstrated that UBF trimethylation at K232/254 by ESET deregulates rDNA transcription in a cell model of Huntington's disease. Together, our findings show that a novel epigenetic modification of UBF is linked to impaired rDNA transcription and nucleolar chromatin remodeling, which may play key roles in the pathogenesis of neurodegeneration.

Figure 1.

ESET is localized in the nucleolus of a cell line model of neurodegeneration. Increased levels of ESET (n = 5) (A) and H3K9me3 (n = 3) (B) were found in STHdh^Q111/111 (Q111) cells in comparison with STHdh^Q7/7 (Q7) cells. Significantly different from control at *P < 0.05; **P < 0.005. (C) The level of ESET was elevated in the nucleolar fraction in where UBF, a nucleolar transcription factor, is mainly found. The purity of subcellular fraction was determined by specific antibody as follows: Lamin B, a nucleus marker; Fibrillarin, a nucleolus marker. (D) The immunoreactivity of ESET was localized in the nucleolus of Q111 cells in comparison with Q7 cells. Scale bar: 10 µm. (E) The line measurement of ESET and UBF immunofluorescence signals showed their co-localization in the nucleolus of Q111 cells but not in Q7 cells. (F) Analysis of the co-localization of UBF with ESET using deconvoluted and 3D reconstructed confocal images. The 3D reconstructed orthoslice image confirms that ESET is co-localized in the peripheral regions of UBF foci in Q111 cells. White arrows indicate DAPI-free hollow regions (nucleolar compartments). Scale bar: 5 µm.

Figure 2.

UBF interacts with ESET and its methylation is modulated by the methyltransferase activity of ESET. (A) UBF interacts with ESET in Q7 and Q111 cells. Cell lysates were immunoprecipitated with anti-UBF antibody and subsequently the blot was probed with anti-ESET antibody. The same blot was stripped and reprobed with anti-UBF antibody. (B) ESET was associated with UBF. (C) A scheme of UBF deletion constructs. Plasmids encoding Flag-UBF and its deletion mutants were transiently transfected in striatal (Q7) cells. The lysates were immunoprecipitated with anti-Flag antibody and the blot was probed with anti-ESET antibody. (D) Schematic representation of ESET functional domains. Plasmids encoding Myc-ESET and its mutants were transfected into Q7 cells. The lysates were immunoprecipitated with anti-Myc antibody and blots were probed with anti-UBF antibody. (E) The level of Tri-Met UBF was increased in Q111 HD cells in comparison with Q7 control cells. The UBF was immunoprecipitated and the blot was probed with anti-Tri-Met-Lys antibody (n = 3). (F) The deletion of SET domain in ESET (ESET–dSET) abrogated the methylation of UBF (n = 3). (G) Knockdown of ESET by shRNA reduced the methylation of UBF (n = 3). ESET (F) and UBF blots (G) were obtained from duplicate gels.

Figure 3.

UBF is methylated at K232/K254 residues by ESET and the methylation status of UBF contributes to the transcriptional activity of rDNA. (A) Liquid chromatography-tandem mass spectrometry analysis identified the methylation site of UBF at K232 and K254 by ESET. The XIC of the trimethylated K232 peptide (DSLGK#QWSQLSDK, m/z = 767.49, doubly change) and tri-methylated K254 peptide (K#EYEEIMR, m/z = 569.94, doubly change) were extracted within the narrow m/z range (m/z = 767.48 to 767.50 in DSLGK#QWSQLSDK and m/z = 569.93 to 569.95 in K#EYEEIMR) using Xcalibur software (Thermo). Representative MS/MS spectra of trimethylated peptides (DSLGK#QWSQLSDK and K#EYEEIMR) are shown in right panels. Fragment ions detected in our study are labeled. The methylation site is marked with a sharp. The SEQUEST matching scores (Xcorr and deltaCN) of peptides were also shown. (B) Mutations at K232R, K254R and K232/254R of GST–UBF–HMG2 domain abrogated the trimethylation by ESET in vitro. (C) Ectopic expression of UBF methylation site mutants (K232A, K254A and K232/254A) reduced the methylation of UBF in intact cells. pCMV-Flag-UBFs (WT, K232A, K254A and K232/254A) were transiently transfected, immunoprecipitated with anti-Flag antibody and blotted with anti-Tri-Met-Lys antibody. The whole blots of Tri-Met Lys in (B) and Tri-Met UBF in (C) are presented in the Supplementary Figure S8. (D) The methylation site mutant of UBF (K232/254A double mutant, DM) resulted in the recovery of rDNA transcriptional activity in response to ESET (n = 5). The methylation site mutant UBF [mtUBF (K232/K254A)] restored 18S (E) (n = 5) and 28S (F) (n = 3) rRNA levels that were decreased by ESET. Double asterisk represents significantly different from control at P < 0.005; Hash represents significantly different from UBF at P < 0.05; Diamond represents Ssignificantly different from UBF with ESET at P < 0.05.

Figure 4.

The level of trimethylated UBF (Tri-Met UBF) is regulated by ESET. (A) The immunoreactivity of Myc-tagged ESET and H3K9me3 was increased by doxycycline (Doxy). (B) The protein level of Myc-tagged ESET was robustly induced by Doxy (n = 3). (C) The increased levels of ESET and TMH3K9 were found in nuclear and nucleolar fractions in response to Doxy. The purity of subcellular fraction was determined by specific antibody as follows: Lamin B, a nucleus marker; Fibrillarin, a nucleolus marker. (D) The induction of ESET by doxycycline for the indicated period of time increased the expression trimethylated UBF in a time-dependent manner. (E) Cells were treated with doxycycline for 36 h (ESET ON) and then cells were washed of doxycycline and switched to the normal fresh media for 36 h (ESET OFF). The level of Tri-Met UBF was increased after 36 h of the ESET ON condition and was decreased to the basal levels after 36 h of the ESET OFF condition and vice versa. This change was depended on the induced or reduced of ESET. The whole blot of Tri-Met UBF in (E) is presented in the Supplementary Figure S8. (F) H3K9me3 immunoreactivity was markedly induced in ESET ON cells compared with ESET OFF cells. Scale bar: 10 µm.

Figure 5.

ESET modulates the occupancy of UBF to rDNA and the expression of rRNA. (A) Positions of qRT–PCR primer set for detecting UBF occupancy in the promoter of mouse rDNA (upper panel). Quantitative ChIP analysis of UBF occupancy in the mouse rRNA gene showed that the UBF binding to rDNA is dependent on ESET induction (bottom panel). (B) In situ transcription assay of 5′-FU incorporation into nucleolar rRNA showed the reduction of rRNA expression in ESET ON cells compared with control and ESET OFF cells (upper panel). The nucleus was counterstained with DAPI. The density (pixels) of BrdU immunoreactivity was averaged by counting each 60 cells in four areas. (C) Time-lapse microscopic analysis showed condensation of nucleolar chromatin in a UBF methylation-dependent manner during the ESET ON condition. (D) UBF methylation by ESET leads to reduced rRNA expression. Q7 cells were transiently transfected with UBF and mtUBF (K232/254A) or with ESET. UBF foci were increased in size by ESET (right upper panel). Mutant UBF methylation restored rDNA transcription compared with WT UBF (right bottom panel). The density of BrdU immunoreactivity was averaged by counting each 30 cells in 10 areas. Double asterisk represents significantly different from control cells at P < 0.005; hash represents significantly different from ESET ON cells at P < 0.05. Scale bar: 10 µm. ENH, enhancer; UCE, upstream control element; CORE, core region; ETS, external transcribed spacer.

Figure 6.

Methylation of UBF by ESET increases the condensation of UBF-mediated nucleosomal structure and the plasticity of chromatin in the nucleolus. (A) AFM analysis and representative 3D topography images showed a change in UBF-dependent nucleosomal structure in ESET ON cells compared with ESET OFF cells (middle panel). Single-molecule line scans showed that the height and distance of UBF-associated nucleosomes are robustly increased during the ESET ON condition (bottom panel). (B) A scheme illustrating that ESET modulates UBF-mediated chromatin remodeling and rDNA transcription in both the normal and the ESET ON conditions, similar to HD.