Figure 2.
Quiescent cells regulate centromere behavior through a transcriptional program. (A) Volcano plot comparing mRNA abundances in quiescent and cycling RPE1 cells as measured by RNA sequencing. Centromere components are highlighted in red. A P value cutoff was imposed at P = 6.84E−305 for genes with P values of 0. Genes with low read counts (total counts <50) were excluded. (B) Graph showing the fold change in mRNA abundance between cycling cells and cells in quiescence for 7 days for the indicated centromere component as quantified by qPCR. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (C) Graph showing mRNA abundance for centromere components over time as cells enter quiescence. Fold change is calculated for each quiescent time point by dividing by cycling value. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (D) Graph showing CENPT mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (E) Graph showing CENPI mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (F) Graph showing CENPO mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (G) Graph showing CENPL mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (H) Graph showing CENPC mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (I) Representative immunofluorescence images of cells ectopically expressing GFP-CENP-N in cycling conditions or quiescence. Cells were stained for GFP (GFP-CENP-N) and anti-centromere antibody (ACA). Scale bar = 5 µm. (J) Graph showing GFP-CENP-N centromere intensity level in cycling and quiescent cells. Results were aggregated for two GFP-CENP-N–overexpressing cell line clones. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue and purple points represent average of each replicate for each clone. Points were aggregated from three replicates for each. n = 398 and 438 cells for cycling and quiescent, respectively. P = 0.0003. *** represents P < 0.001, using unpaired t test with Welch’s correction. (K) Graph showing the correlations in the gene expression profiles of various centromere protein genes and FoxM1 across samples from the Expression Project for Oncology (expO) data set (GEO accession number GSE2109). Correlations are ordered from largest to smallest and obtained from Thiru et al. (2014). (L) Graph showing FoxM1 binding to centromere protein gene promoters. ChIP-seq signal (ENCODE Project Consortium, 2012) was analyzed for FoxM1 compared with a control IP for regions encompassing 1 kb upstream and 100 base pairs downstream of the transcriptional start site for kinetochore genes. Fold enrichment values were obtained from Thiru et al. (2014). Refer to the image caption for details. Panel A: A volcano plot compares m R N A abundances in quiescent and cycling R P E 1 cells, highlighting centromere components in red. The horizontal axis represents the log 2 fold change, and the vertical axis represents the negative log 10 p-value. Panel B: A bar graph shows the fold change in m R N A abundance for various centromere components after 7 days in quiescence, quantified by q P C R. The vertical axis represents normalized abundance, and bars indicate mean and standard deviation. Panel C: A line graph displays m R N A abundance for centromere components over time as cells enter quiescence, with fold change calculated by dividing quiescent values by cycling values. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel D: A line graph shows C E N P T m R N A abundance over time after adding Actinomycin D in cycling and quiescent cells. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel E: A line graph shows C E N P I m R N A abundance over time after adding Actinomycin D in cycling and quiescent cells. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel F: A line graph shows C E N P O m R N A abundance over time after adding Actinomycin D in cycling and quiescent cells. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel G: A line graph shows C E N P L m R N A abundance over time after adding Actinomycin D in cycling and quiescent cells. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel H: A line graph shows C E N P C m R N A abundance over time after adding Actinomycin D in cycling and quiescent cells. The vertical axis represents normalized abundance, and lines indicate mean and standard deviation. Panel I: Immunofluorescence images show G F P-C E N P-N overexpressing cells in cycling conditions or quiescence, stained for G F P and anti-centromere antibody. Panel J: A scatter plot shows G F P-C E N P-N centromere intensity levels in cycling and quiescent cells, with points representing average intensity values normalized to cycling. Panel K: A bar graph shows correlations in gene expression profiles of various centromere protein genes and F o x M 1 across samples from the Expression Project for Oncology data set. Panel L: A bar graph shows F o x M 1 binding to centromere protein gene promoters, with fold enrichment values obtained from C h I P-seq signal analysis. All data is approximate.

Quiescent cells regulate centromere behavior through a transcriptional program. (A) Volcano plot comparing mRNA abundances in quiescent and cycling RPE1 cells as measured by RNA sequencing. Centromere components are highlighted in red. A P value cutoff was imposed at P = 6.84E−305 for genes with P values of 0. Genes with low read counts (total counts <50) were excluded. (B) Graph showing the fold change in mRNA abundance between cycling cells and cells in quiescence for 7 days for the indicated centromere component as quantified by qPCR. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (C) Graph showing mRNA abundance for centromere components over time as cells enter quiescence. Fold change is calculated for each quiescent time point by dividing by cycling value. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (D) Graph showing CENPT mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (E) Graph showing CENPI mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (F) Graph showing CENPO mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (G) Graph showing CENPL mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (H) Graph showing CENPC mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Fold change is calculated for each ActD time point by dividing by the untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (I) Representative immunofluorescence images of cells ectopically expressing GFP-CENP-N in cycling conditions or quiescence. Cells were stained for GFP (GFP-CENP-N) and anti-centromere antibody (ACA). Scale bar = 5 µm. (J) Graph showing GFP-CENP-N centromere intensity level in cycling and quiescent cells. Results were aggregated for two GFP-CENP-N–overexpressing cell line clones. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue and purple points represent average of each replicate for each clone. Points were aggregated from three replicates for each. n = 398 and 438 cells for cycling and quiescent, respectively. P = 0.0003. *** represents P < 0.001, using unpaired t test with Welch’s correction. (K) Graph showing the correlations in the gene expression profiles of various centromere protein genes and FoxM1 across samples from the Expression Project for Oncology (expO) data set (GEO accession number GSE2109). Correlations are ordered from largest to smallest and obtained from Thiru et al. (2014). (L) Graph showing FoxM1 binding to centromere protein gene promoters. ChIP-seq signal (ENCODE Project Consortium, 2012) was analyzed for FoxM1 compared with a control IP for regions encompassing 1 kb upstream and 100 base pairs downstream of the transcriptional start site for kinetochore genes. Fold enrichment values were obtained from Thiru et al. (2014).

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