Mutations Disrupting Histone Methylation Have Different Effects on Replication Timing in <i>S. pombe</i> Centromere

<div><p>The fission yeast pericentromere comprises repetitive sequence elements packaged into heterchromatin marked by histone H3K9 methylation and Swi6 binding. Transient disruption of Swi6 during S phase allows a period of RNA synthesis which programs the RNAi machinery to maintain histone methylation. However, Swi6 is also required for early replication timing. We show that not only Swi6 but also the chromodomain protein Chp1 are delocalized during S phase. Different from loss of <i>swi6</i>, mutations that disrupt histone methylation in the centromere, <i>chp1</i>Δ and <i>clr4</i>Δ, undergo early DNA replication. However, timing is modestly delayed in RNAi mutants <i>dcr1</i>Δ or <i>rdp1</i>Δ, while <i>hrr1</i>Δ mutants resemble <i>swi6</i>Δ in their replication delay. Finally, we show that recruitment of RNA polymerase II in the centromere occurs independently of replication. These different effects indicate that replication timing is not simply linked to histone methylation.</p></div

The centromere replicates early in <i>clr4</i>Δ or <i>chp1</i>Δ mutants.

<p>A, the structure of the three <i>S. pombe</i> centromeres. Repetitive sequences <i>dg</i> and <i>dh</i> in the outer repeats (<i>otr</i>) are present in slightly different organization in each centromere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061464#pone.0061464-Tran1" target="_blank">[66]</a>. The length of <i>dg</i> or <i>dh</i> is around 4-6 kb. B, scheme of the experimental protocol. <i>cdc25-22</i> mutants were shifted to 36°C for 4 hours. One hour prior to release to 25°C, 10 mM of HU was added so that only early origins fire. Upon release, 100 µg/ml BrdU was added to label new DNA synthesis. C, incorporation of BrdU in the <i>dg</i> region was detected by BrdU enrichment, which was calculated by the ratio of IP versus Input by semi-quantitative PCR using primers #1536/1537 (dg). Three independent experiments were performed. Asterisks mark samples with BrdU signal significantly higher than the WT at 90 min with p<0.05 (Student's T test). The quality of synchronization is determined by flow cytomertry. There is no significant different among WT, <i>chp1</i>Δ and <i>clr4</i>Δ (Figure S1A).</p

Swi6 and Chp1 each delocalize from the centromere between M and S phase.

<p>Asynchronously growing cells containing CFP-Cnp1 Sad1-DsRed (FY4229), GFP-Swi6 Sad1-DsRed (FY3665), or Chp1-GFP Sad1-DsRed (FY5911) were imaged every three minutes and the images were projected as described in materials and methods. A, quantitation of the data. The first frame in which spindle pole body duplication was observed was assigned as “0”. The first frame in which re-association of the GFP marked protein with the SPB occurred, and was maintained for at least three frames, was recorded. The re-association timing of CFP-Cnp1 (green), GFP-Swi6 (purple), or Chp1-GFP (black) with Sad1-DsRed is summarized in this distribution. Y axis, number of cells; X axis, time (minutes) after duplication of Sad1-DsRed, which is used to mark the beginning of M phase. Representative frames of GFP-Swi6, or Chp1-GFP are provided here B-C, respectively; selected original movies are provided in Movies S1, S2, S3. Arrows show cells with delocalization of the GFP signal from the SPB.</p

Replication timing and polymerases recruitment in wild type and <i>swi6</i>Δ.

<p>Cells were synchronized by <i>cdc25-22</i> block and release. Two independent experiments were performed and the representative result is presented. The signals were determined by quantitative real-time PCR. A, position of PCR probes used in this experiment. B–C, recruitment of DNA polymerase alpha (blue line), RNA polymerase II (green line) and incorporation of BrdU (red line) in euchromatin and the centromere, respectively. Left Y axis, DNA polymerase alpha or RNA polymerase II enrichment; right Y axis, BrdU enrichment. Red arrow, early replication time-point; blue arrow, late replication time-point. Primers: #1257/1258 (ars2004), #1265/1266 (non-ars), #875/876 (AT2080), #1628/1629 (dg), #1183/1184 (dh), and #879/880 (cnt2).</p

Centromere replication is delayed in RNAi mutants.

<p>A, schematic showing the experimental procedure. Cells were arrested in G1 by nitrogen depletion and released into the cell cycle by refeeding. BrdU and HU were added at 1.5 hours after release. B-D, BrdU ChIP in different mutants at the early origin <i>ars2004</i> (B), and the centromere repeats <i>dg</i> (C), and <i>dh</i> (D). BrdU enrichment was calculated by the ratio of IP versus Input by semi-quantitative PCR and at least two independent experiments were performed. Each mutant compared to its 0 timepoint. Asterisks mark samples with BrdU signal significantly higher than the WT at 6 h timepoint with p<0.05 (Student's T test). Primers: #1041/1042(dg), #1033/1034 (dh), and #1257/1258 (ars2004).</p

ER-α and ER-β bind to the amino-terminal and carboxyl-terminal domains of FOXO3a, respectively

Glutathione-S-transferase (GST) – pull down assays. Whole cell lysates from 293T cells were incubated with the GST-forkhead box class O (FOXO)3a (GST-FO [amino acids 1 to 300] and GST-FO [amino acids 301 to 673]) fusion proteins as indicated and GST alone (negative control), and analyzed by SDS-PAGE and immunoblotting with an antibody (Ab) against estrogen receptor (ER)-α or ER-β (upper panels) and an anti-GST Ab (lower panel) as protein controls. FOXO3a downregulates FOXM1. Immunoblotting (IB) analyses for endogenous FOXO3a, forkhead box M1 (FOXM1), and β-actin (loading control) protein expression in MCF7-FO10 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 wt and MCF7-C12) cells were performed with specific antibodies antibodies as indicated. MCF7-d8_pa cells (pooled clones of MCF-7 FOXO3a-knockdown derivatives) were established with retroviruses expressing small hairpin RNA against human FOXO3a. The expression levels of FOXO3a and p27Kip1 in MCF-7 wild-type (wt) and MCF7-d8_pa cells were determined by IB with specific Abs against FOXO3a or p27Kip1 or β-actin (loading control). Silencing endogenous FOXO3a in MCF-7 cells promoted tumorigenesis . The tumor growth rates of control group MCF-7 wt and knockdown group MCF7-d8-pa were determined after injection of cells (2 × 10cells/mouse) as indicated into the mammary fat pads of female athymic mice not given supplemental 17β-estradiol (E2; indicated as – E2). Data are expressed as means and standard deviations from two experiments with five mice in each group. *< 0.01 between MCF7-d8-pa group versus control MCF-7 wt group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

FOXO3a and FOXO1a inhibit the transactivation activities of ER-α and ER-β

293T cells were co-transfected with estrogen receptor (ER)-responsive element (ERE)-luc (firefly luciferase [luc] reporter containing EREs), pRL-TK (renilla luc as a transfection control for normalization), ER-α (panel a) or ER-β (panel b), and forkhead box class O (FOXO)3a plus IκB kinase (IKK)-β or an empty vector (control) as indicated. Total lysates of the transfected cells were prepared and subjected to luc assays. Total lysates of 293T cells were co-transfected with ERE-luc, pRL-TK, ER-α (panel c) or ER-β (panel d), and FOXO1a plus IKK-β or an empty vector as indicated and subjected to luc assays. All cells were cultured in the presence of 17β-estradiol (E2). The relative reporter luc activity was normalized with pRL-TK. Data are expressed means and standard deviations from three repeated experiments, which were performed independently. *< 0.05 between FOXO (FOXO3a or FOXO1a) minus IKK-β (lane 3) versus FOXO plus IKK-β (lane 4).<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

FOXO3a regulates expression of ER target genes and CDK inhibitors, and induces apoptosis in MCF-7

Ectopic expression of Forkhead box class O (FOXO)3a reduces the expression of some estrogen receptor (ER)-regulated genes and enhances the expression of cyclin-dependent kinase (CDK) inhibitors in MCF7-FO cells in the presence of 17β-estradiol (E2). Immunoblotting (IB) analyses for HA-FOXO3a, endogenous FOXO3a, p27Kip1, p21Cip1, p57Kip2, cyclin D, cyclin E, cathepsin D, progesterone receptor (PgR), ER-α, and ER-β protein expression in MCF7-FO33 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 and MCF7-C5) cells were performed with specific antibodies, as indicated. Equal loading was confirmed by the same IB analysis with antibodies against β-actin or β-tubulin. MDA-MB-453 (MDA-453, ER-negative) cells were transfected with either FOXO3a or an empty pCDNA3.1 vector (control), as indicated. Total lysates of the transfected cells were prepared and subjected to SDS-PAGE followed by IB analysis with the indicated antibodies.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

Ectopic expression of FOXO3a in estrogen-dependent breast cancer cells suppresses breast tumor development

Forkhead box class O (FOXO)3a suppresses estrogen receptor (ER)-positive breast tumor development in a mouse model of breast cancer. The MCF7-FO pooled cell lines and the control MCF7-C pooled cell lines were injected (2 × 10cells/mouse) into the mammary fat pads of female athymic mice given supplementary 17β-estradiol (E2), as described in Materials and methods. Growth curves of tumor size are the means of the MCF7-FO pooled cell lines (designated MCF7-FO) and the control MCF7-C pooled cell lines (designated MCF7-C); error bars indicate standard deviation from three experiments. *< 0.05 between control MCF7-C group versus MCF7-FO group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

Ectopic expression of FOXO3a in estrogen-dependent breast cancer cells suppresses cell proliferation in cell culture

Growth of forkhead box class O (FOXO)3a over-expressing MCF7-FO (MCF7-FO10, MCF7-FO33, and MCF7-FO41) cells and control (MCF7-C4, MCF7-C5, and MCF7-C12) cells in the absence of 17β-estradiol (E2) was determined by counting trypan-blue stained cells with a hemocytometer. Growth curves are the means of the three MCF7-FO cell lines (MCF7-FO10, MCF7-FO33, and MCF7-FO41) and the three control cell lines (MCF7-C4, MCF7-C5, and MCF7-C12); error bars indicate standard deviation from three experiments. Growth of the same sets of cells in the presence of E2 (1 nmol/l). Growth curves are the means of the three MCF7-FO cell lines (designated MCF7-FO average) and the three control cell lines (designated MCF7-C average); error bars indicate standard deviaton from three experiments. *< 0.05 between control MCF7-C group versus MCF7-FO group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p