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, 122 (Pt 4), 489-98

Involvement of SIRT7 in Resumption of rDNA Transcription at the Exit From Mitosis

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Involvement of SIRT7 in Resumption of rDNA Transcription at the Exit From Mitosis

Alice Grob et al. J Cell Sci.

Abstract

Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.

Figures

Fig. 1.
Fig. 1.
Endogenous SIRT7 co-localizes with UBF in interphase and mitotic HeLa cells. (A) Immunodetection of SIRT7, UBF and rDNA transcription sites was obtained in fixed interphase HeLa cells using anti-SIRT7-C-terminal (b,f), anti-UBF (c) and anti-BrdU (g) antibodies. Images were obtained using confocal laser microscopy. In merged images (d,h), yellow labeling indicates co-localization of green (SIRT7) and red (UBF or BrdU) signals. (B) SIRT7 signal obtained by immunofluorescence in control HeLa cells (a,b) is no longer detected after SIRT7 knockdown (c,d; arrow). (C) HeLa cells were synchronized in mitosis. Mitotic stages were determined on Z projections of focal planes by DAPI staining (a,e). SIRT7 (arrowheads b,f) and UBF (c,g) were immunodetected in mitotic HeLa cells. Immunofluorescence labelings were merged (d,h). (D) SIRT7 and UBF signal intensity was quantified in 30 NORs for each stage of mitosis. The ratios of the mean intensity value were quantified (P, 0.24±0.10; M, 0.11±0.03; A, 0.16±0.06; T, 0.28±0.06; T', 0.94±0.22). Bars, 10 μm. A, anaphase; M, metaphase; P, prophase; T, early telophase; T', late telophase.
Fig. 2.
Fig. 2.
SIRT7 is recruited to pseudo-NORs via direct interaction with UBF. (A) GST and GST-SIRT7 pull-down assays were performed using whole HeLa cell extracts. 1% of extract used for each pull-down assay (Input), 5% of the proteins eluted after GST-SIRT7 pull-down assays and 5% of the proteins eluted after GST pull-down assays were submitted to 10% SDS-PAGE. Immunoblotting was performed using anti-PAF49, anti-PAF53, anti-RPA116/135, anti-RPA43, anti-TIF-1A/Rrn3, anti-UBF, anti-TTF-1 and anti-TBP antibodies. (B) GST and GST-SIRT7 pull-down assays were performed using 1 μg purified human UBF1 or UBF2. (C) The pseudo-NOR containing cell line 3D-1 was stained with anti-SIRT7-C-terminal and anti-UBF antibodies (a-d). Red and green labelings were merged (d). Pseudo-NORs are identified by arrowheads. Bar, 10 μm.
Fig. 3.
Fig. 3.
Increase in SIRT7 signal corresponds to increased reactivity of antibodies to the SIRT7 C-terminal region. (A) In nocodazole-arrested HeLa cells treated (d-f) or not (a-c) with roscovitine (150 μM for 1 hour), SIRT7 and UBF were immunodetected using anti-SIRT7-C-terminal (c,f) and anti-UBF (b,e) antibodies. DNA was stained by DAPI and merged with red and green labelings (a,d). (B) SIRT7 signal intensity was quantified in 10 cells. The means of intensity value were quantified (Control: 0.25±0.06, Roscovitine: 0.83±0.15). (C) Cytoplasmic (lanes 1 and 3) and chromosome (lanes 2 and 4) fractions prepared from nocodazole-arrested HeLa cells treated (lanes 3 and 4) or not (lanes 1 and 2) with roscovitine were submitted to 10% SDS-PAGE. Immunoblotting was performed using anti-UBF, anti-SIRT7-N-terminal and anti-fibrillarin antibodies. (D) HeLa cells were synchronized in mitosis and mitotic stages were determined by DAPI staining (a,d,g). In immunofluorescence assays, SIRT7 and UBF were detected using anti-SIRT7-N-terminal (c,f,i) and anti-UBF (b,e,h) antibodies, respectively. Immunofluorescence labelings were merged with DAPI (a,d,g). Bars, 10 μm.
Fig. 4.
Fig. 4.
Increased reactivity of antibodies to the SIRT7 C-terminal region occurs before, and independently of, onset of rDNA transcription. (A) Surrounded mitosis stages (M, T, T') of synchronized HeLa cells were determined on Z projections of focal planes by DAPI staining (a,e). SIRT7 and rDNA transcription sites were detected in fixed cells using respectively anti-SIRT7-C-terminal (b,f) and anti-BrdU (c,g) antibodies. Red and green labelings were merged (d,h). (B) Immunodetection of SIRT7 (c,h) and rDNA transcription sites (d,i) was performed in early G1 fixed cells in the absence (a-e) or presence (f-j) of AMD (0.1 μg/ml for 2 hours). Early G1 cells (arrows) were identified by phase-contrast (a,f) and DAPI staining (b,g). Arrowheads indicate metaphase cells. Immunofluorescence labelings were merged (e,j). Bars, 10 μm.
Fig. 5.
Fig. 5.
Inhibition of CDK1-cyclin B induces increased reactivity of antibodies to the SIRT7 C-terminal region by SIRT7 dephosphorylation. (A) SIRT7 and rDNA transcription sites were immunodetected using anti-SIRT7-C-terminal (b,f,j,n) and anti-BrdU (c,g,k,o) antibodies in prometaphase mitotic HeLa cells, in the absence (a-d) or presence (e-h) of roscovitine (150 μM for 1 hour). Mitotic HeLa cells were also treated with AMD (0.1 μg/ml, i-l) or okadaic acid (0.5 μM, m-p) for 1 hour before addition of 150 μM roscovitine for 1 hour. Red and green labelings were merged (d,h,l,p). (B) Whole cell extracts from unsynchronized (U) or nocodazole-arrested mitotic (M) HeLa cells were submitted to 10% SDS-PAGE. Immunodetection of SIRT7 was performed using anti-SIRT7-N-terminal antibodies. (C) Whole cell extracts prepared from unsynchronized HeLa cells (Unsyn, lane 1) and from nocodazole-arrested mitotic HeLa cells (lanes 2-4) treated with okadaic acid (0.5 μM for 1 hour) (lane 4) or not (lane 3) before addition of roscovitine (150 μM for 1 hour) (lanes 3 and 4) were submitted to 7.5% Phos-tag SDS-PAGE. Immunodetection of SIRT7 was performed using anti-SIRT7-N-terminal antibodies. (D) SIRT7 presents a consensus CDK1 phosphorylation site (ATPLR) at amino acids 344-348 (red box). Blue boxes indicate the peptides against which anti-SIRT7-N-terminal antibodies (amino acids 32-51) and anti-SIRT7-C-terminal antibodies (amino acids 384–400) are directed. Bar, 10 μm.
Fig. 6.
Fig. 6.
SIRT7 is involved in resumption of rDNA transcription at the exit from mitosis. (A) BrUTP incorporation in living cells was detected using anti-BrdU antibodies in interphase HeLa cells in the absence (a-b) or presence of AMD (0.1 μg/ml, c-d, or 4 μg/ml, e-f, for 2 hours) or sirtinol (100 μM for 6 hours, g-h). (B) Knockdown of SIRT1 and SIRT7 was analyzed by 10% SDS-PAGE and immunoblotting using anti-SIRT1 and anti-SIRT7-N-terminal antibodies. (C) SIRT1 and BrUTP incorporation were detected using anti-SIRT1 (b,e) and anti-BrdU (c,f) antibodies, respectively, in control (a-c) or SIRT1-depleted (d-f) HeLa cells. (D) SIRT7 and BrUTP incorporation were detected using anti-SIRT7 (b,e) and anti-BrdU (c,f) antibodies, respectively, in control (a-c) or SIRT7-depleted (d-f) HeLa cells. (E) SIRT7 and rDNA transcription sites were immunodetected using anti-SIRT7-C-terminal (b,f,j) and anti-BrdU (c,g,k) antibodies in prometaphase mitotic HeLa cells, in the absence (a-d) or presence (e-h) of roscovitine (150 μM for 1 hour). Mitotic HeLa cells were also treated with 100 μM sirtinol for 2 hours before addition of 150 μM roscovitine for 1 hour (i-l). Red and green labelings were merged (d,h,l). Bars, 10 μm.

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