Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.

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.

Expression of topoisomerase IIalpha, Ki-67, proliferating cell nuclear antigen, p53, and argyrophilic nucleolar organizer regions in vulvar squamous lesions.

OBJECTIVE: It was the aim of this study to investigate the expression of topoisomerase IIalpha (topo IIalpha), Ki-67, proliferating cell nuclear antigen (PCNA), p53, and argyrophilic nucleolar organizer region (AgNOR) staining in normal vulvar epithelia (NE, N = 10), vulvar condylomas (VC, N = 24), vulvar intraepithelial neoplasia (VIN, N = 26), as well as squamous cell carcinomas (SCC, N = 22) of the vulva. METHODS: Formalin-fixed, paraffin-embedded archival tissue sections were immunostained with monoclonal antibodies against topo IIalpha, p53, and PCNA, as well as an affinity-isolated prediluted ready-to-use Ki-67 antibody using a standard immunohistochemical method, and stained with a colloid silver solution for AgNORs. Immunostaining was quantitated by determining the percentage of positively staining nuclei in each sample to express the labeling indices (LIs) by counting the immunoreactive nuclei in 1000 epithelial cells per case for each antibody. In each specimen 200 nuclei were examined using a x100 oil emersion lens, and the mean number of AgNORs per nucleus (AC) was calculated. RESULTS: The LIs for topo IIalpha, Ki-67, and PCNA as well as ACs increased stepwise from NE to VCs, VIN lesions, and SCCs. In contrast to PCNA LIs and ACs, a consistent correlation in all four groups was found for Ki-67 and topo IIalpha, suggesting that the latter is a proliferation-associated marker in these tissues. p53 expression was seen 8.3% of VCs, 30.8% of VIN lesions, and 54.45% of SCCs. p53 LIs were not correlated with LIs for topo IIalpha or Ki-67 in SCCs. The LIs for topo IIalpha, Ki-67, PCNA, p53, and ACs were not related to tumor progression, FIGO stage, or tumor grade in SCCs. CONCLUSIONS: This study presents topo IIalpha and Ki-67 as useful proliferation-associated markers of vulvar epithelia.

The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery.

The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.

Is DNA topoisomerase II beta a nucleolar protein?

We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo II alpha was overall similar doing interphase and mitosis to that previously reported, regardless of antibody and of sample preparation. On the other land, the interphase distribution of Topo II beta was quite variable, depending both on the antibody and on the method used to prepare the sample. Our interpretation of the data is that, like Topo II alpha, Topo II beta is primarily a nucleoplasmic protein, but that unlike Topo II alpha, small amounts are also associated with intranucleolar chromatin.