TopBP1 Controls Cell-Cycle Progression at G1 and S (II)

Human TopBP1 was initially characterised to function in DNA damage response and DNA replication and to be essential for cell survival (Mäkiniemi et al., 2001; Yamane et al., 2002). Identifica-tion of TopBP1 as an essential component for activaIdentifica-tion of ATR was a big leap in understanding the function of both ATR and TopBP1 (Kumagai et al., 2006).

The main target in ATR signalling is Chk1 kinase, which is re-sponsible for the global effects of ATR activation (Cimprich &

Figure 3. UBF interacts with TopBP1 in vivo and is required for its focal localisation.

(A) Immunoprecipitation (IP) with anti-eGFP or anti-UBF antibodies from nuclear ex-tracts of cells that were induced to express eGFP (G) or eGFP-TopBP1 (T) for 24 h prior to assay. Input is the cellular extract before immunoprecipitation. (B) eGFP-TopBP1 cells transfected with UBF siRNA #1 or #2 or unspecific siRNA (NEG siRNA) or left untransfected (Control). Expression of eGFP-TopBP1 was induced for 24 h before fixing the cells. DNA was stained with Hoechst 33258. Scale bar is 10 µm. (C) Immunoblot of the whole-cell extracts from B. (D) Mean number (± standard deviation) of TopBP1 foci per cell from B. Results are means of three independent experiments (more than 70 nu-clei were counted in each experiment).

formed. Transcriptionally inactive pseudo-NORs appear in in-terphase nuclei as tightly packed bodies, similar to ActD-induced nucleolar caps. It has been proposed that these nucleolar caps and pseudo-NOR bodies are related phenomena that depend on UBF (Prieto & McStay, 2008). Interestingly, we found that UBF and

TopBP1 interacted in cells (Figure 3). UBF and eGFP-TopBP1 were reciprocally co-immunoprecipitated from cells (Figure 3A). Depletion of UBF by siRNA reduced the focal locali-sation of TopBP1, suggesting that UBF is involved in the nucleo-lar segregation (Figure 3B–D). The ability of TopBP1–ATR to in-duce nucleolar segregation suggests a role in remodelling the chromatin structure from an active open state to a condensed and transcriptionally repressed state.

Surprisingly, while ActD induced robust p53 activation, in-crease in p21 levels, and replicative arrest lasting for several days, high levels of TopBP1 resulted only in local accumulation of acti-vated p53 and a modest increase in p53 levels, but no replication arrest (I-Fig 7). However, the lack of p53 checkpoint arrest under high levels of TopBP1 is consistent with the negative effect of TopBP1 on p53 transcriptional targets (Liu et al., 2009).

Taken together, our results show that the TopBP1–ATR path-way can activate nucleolar stress response. Others have reported inhibition of RNA pol I through different stress sensors leading to nucleolar stress response (Mayer et al., 2005; Kruhlak et al., 2007; Larsen et al., 2014; van Sluis & McStay, 2015). These findings add to the evidence that the nucleolar stress response pathway is efficiently exploited by different cellular stress signalling path-ways through a common mechanism by inhibition of transcrip-tion by RNA pol I.

3.2 TOPBP1 CONTROLS CELL-CYCLE PROGRESSION AT G1 AND S (II)

Human TopBP1 was initially characterised to function in DNA damage response and DNA replication and to be essential for cell survival (Mäkiniemi et al., 2001; Yamane et al., 2002). Identifica-tion of TopBP1 as an essential component for activaIdentifica-tion of ATR was a big leap in understanding the function of both ATR and TopBP1 (Kumagai et al., 2006).

The main target in ATR signalling is Chk1 kinase, which is re-sponsible for the global effects of ATR activation (Cimprich &

Cortez, 2008). When the replication fork stalls or collapses due to DNA damage or a physical obstacle, ATR and TopBP1 are inde-pendently recruited to the sites of damage. The physical contact among ATR, ATRIP (ATR interacting protein), and TopBP1 leads to signal amplification and activation of Chk1. Chk1 is released from the chromatin and diffused throughout the nucleus, where it inhibits activation of new replication domains through actions on Cdc25 and Wee1 (Sørensen & Syljuåsen, 2012). DNA replica-tion follows a strict temporal and spatial programme, where DNA is replicated within replication factories that include multi-ple replication forks. When replication forks encounter problems, ATR signalling activates dormant origins within the active do-main, but at the same time prevents activation of new replication domains. This signalling mechanism ensures that the DNA repli-cation is completed in active domains and prevents further po-tential damage by inhibiting replication globally. How these local (acting at the site of DNA lesion) and global (genome-wide) ef-fects of ATR are regulated is not well understood.

The ATR activation can be abolished by a single tryptophan to arginine mutation at 1145 (W1145R) in TopBP1 (Kumagai et al., 2006). To better understand the TopBP1-ATR signalling pathway during the cell cycle and in DNA replication, a cell line that con-ditionally expresses the ATR activation defective point mutant of TopBP1 was prepared (TopBP1 W1145R). We found that during DNA replication, TopBP1 W1145R acted dominant-negatively, increasing the number of fired origins and decreasing the dis-tance between activated origins (II-Fig 2). At the same time, rep-lication fork speed was slowed down and the fork symmetry ratio perturbed, indicating defects in elongation of the forks and fork stalling (II-Fig 2). Similar effects have been observed when Chk1 or ATR is inhibited (Shechter et al., 2004; Petermann et al., 2006;

Petermann et al., 2010), and thus our results can directly be at-tributed to the inability of TopBP1 to activate ATR. However, the essential role of TopBP1 in restricting the excess of origin firing has not been previously described.

We also followed TopBP1 W1145R expressing cells in the cell cycle and observed that a large number of cells stopped in the G1

phase (II-Fig 1). This arrest in G1 was accompanied by increase in p21 and p27 cell-cycle inhibitors (II-Fig 1E). When expressed for a prolonged time, TopBP1 W1145R caused the cells to exit from the cell cycle and enter senescence, as shown by the increase in senescence-associated E-galactosidase activity (II-Fig 1F). While the inability of TopBP1 W1145R cells to enter the S phase could be caused by DNA damage inflicted in the previous S phase, it could also mean that TopBP1-ATR signalling is required for entry into the S phase. We tested this by synchronising the cells in the late M phase and releasing them in medium which promoted ex-pression of either WT or W1145R of TopBP1. The exex-pression of TopBP1 W1145R reduced the appearance of cyclin A and phos-phorylated Rb, indicating that the cells were unable to pass the restriction point (II-Fig 3). This inhibitory effect was more clearly demonstrated by including ATR inhibitor in the release medium, suggesting that an intact ATR pathway is required for the cells to proceed in the cell cycle.

Our findings show that TopBP1-induced ATR signalling is re-quired at two independent points during the cell cycle: 1) in the S phase to balance the number of active forks within an active rep-lication domain and 2) in the G1 phase to pass the restriction point (II-Fig 3D).

TopBP1 has previously been suggested to be required for G1 to S phase progression (Jeon et al., 2007). The authors found that inhibition of TopBP1 by siRNA led to up-regulation of p21 and p27, which resulted in down-regulation of cyclin E. This inhibi-tory effect of TopBP1 down-regulation is contradicinhibi-tory to another finding where siRNA-mediated depletion of TopBP1 did not pre-vent entry into the S phase (Kim et al., 2005). It is possible that the TopBP1 checkpoint function is more sensitive to protein levels than DNA replication initiation function.

The current literature indicates opposing roles for TopBP1 in either promotion or inhibition of cell-cycle progression. The strong inhibitory effect on passing the restriction point of ATR-deficient cells suggests that cells monitor the integrity of the ATR pathway before entering a new cell cycle. This could be a way for

Cortez, 2008). When the replication fork stalls or collapses due to DNA damage or a physical obstacle, ATR and TopBP1 are inde-pendently recruited to the sites of damage. The physical contact among ATR, ATRIP (ATR interacting protein), and TopBP1 leads to signal amplification and activation of Chk1. Chk1 is released from the chromatin and diffused throughout the nucleus, where it inhibits activation of new replication domains through actions on Cdc25 and Wee1 (Sørensen & Syljuåsen, 2012). DNA replica-tion follows a strict temporal and spatial programme, where DNA is replicated within replication factories that include multi-ple replication forks. When replication forks encounter problems, ATR signalling activates dormant origins within the active do-main, but at the same time prevents activation of new replication domains. This signalling mechanism ensures that the DNA repli-cation is completed in active domains and prevents further po-tential damage by inhibiting replication globally. How these local (acting at the site of DNA lesion) and global (genome-wide) ef-fects of ATR are regulated is not well understood.

The ATR activation can be abolished by a single tryptophan to arginine mutation at 1145 (W1145R) in TopBP1 (Kumagai et al., 2006). To better understand the TopBP1-ATR signalling pathway during the cell cycle and in DNA replication, a cell line that con-ditionally expresses the ATR activation defective point mutant of TopBP1 was prepared (TopBP1 W1145R). We found that during DNA replication, TopBP1 W1145R acted dominant-negatively, increasing the number of fired origins and decreasing the dis-tance between activated origins (II-Fig 2). At the same time, rep-lication fork speed was slowed down and the fork symmetry ratio perturbed, indicating defects in elongation of the forks and fork stalling (II-Fig 2). Similar effects have been observed when Chk1 or ATR is inhibited (Shechter et al., 2004; Petermann et al., 2006;

Petermann et al., 2010), and thus our results can directly be at-tributed to the inability of TopBP1 to activate ATR. However, the essential role of TopBP1 in restricting the excess of origin firing has not been previously described.

We also followed TopBP1 W1145R expressing cells in the cell cycle and observed that a large number of cells stopped in the G1

phase (II-Fig 1). This arrest in G1 was accompanied by increase in p21 and p27 cell-cycle inhibitors (II-Fig 1E). When expressed for a prolonged time, TopBP1 W1145R caused the cells to exit from the cell cycle and enter senescence, as shown by the increase in senescence-associated E-galactosidase activity (II-Fig 1F). While the inability of TopBP1 W1145R cells to enter the S phase could be caused by DNA damage inflicted in the previous S phase, it could also mean that TopBP1-ATR signalling is required for entry into the S phase. We tested this by synchronising the cells in the late M phase and releasing them in medium which promoted ex-pression of either WT or W1145R of TopBP1. The exex-pression of TopBP1 W1145R reduced the appearance of cyclin A and phos-phorylated Rb, indicating that the cells were unable to pass the restriction point (II-Fig 3). This inhibitory effect was more clearly demonstrated by including ATR inhibitor in the release medium, suggesting that an intact ATR pathway is required for the cells to proceed in the cell cycle.

Our findings show that TopBP1-induced ATR signalling is re-quired at two independent points during the cell cycle: 1) in the S phase to balance the number of active forks within an active rep-lication domain and 2) in the G1 phase to pass the restriction point (II-Fig 3D).

TopBP1 has previously been suggested to be required for G1 to S phase progression (Jeon et al., 2007). The authors found that inhibition of TopBP1 by siRNA led to up-regulation of p21 and p27, which resulted in down-regulation of cyclin E. This inhibi-tory effect of TopBP1 down-regulation is contradicinhibi-tory to another finding where siRNA-mediated depletion of TopBP1 did not pre-vent entry into the S phase (Kim et al., 2005). It is possible that the TopBP1 checkpoint function is more sensitive to protein levels than DNA replication initiation function.

The current literature indicates opposing roles for TopBP1 in either promotion or inhibition of cell-cycle progression. The strong inhibitory effect on passing the restriction point of ATR-deficient cells suggests that cells monitor the integrity of the ATR pathway before entering a new cell cycle. This could be a way for

cells to ensure that the replication phase is possible to complete with minimal damage to the genome.

Single-nucleotide polymorphisms of TopBP1 gene have been linked to increased risk of breast, ovarian and endometrial can-cers (Karppinen et al., 2006; Forma et al., 2013; Forma et al., 2014).

Furthermore, high TopBP1 protein expression levels have been linked with negative prognosis in breast cancer (Liu et al., 2009) and non-small cell lung cancer patients (Wang et al., 2015). These findings, together with the finding of a potentially therapeutic lead compound that blocks TopBP1 function (Chowdhury et al., 2014), provide support for targeting TopBP1 to treat cancer.