Transcription factor Sp1 regulates mitotic chromosome assembly and segregation

Abstract

Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

Keywords: Chromosome passenger complex; Chromosome segregation; Condensin complex I; Mitosis; Sp1; Transcription factor.

Fig. 1

Sp1 localizes to mitotic centromeres. a Top: RPE-1 cells were arrested in metaphase, spread onto a glass slide, and stained for Sp1 and CENP-A. Scale bar = 10 µm. Bottom: Representative chromosome. Scale bar = 1 µm. b Images taken in live RPE1^{TdTomato−CENP−A} cells transfected with Sp1-GFP. Cell cycle phase was scored based on chromosome conformation. Insets are representative cropped images of Sp1-GFP and TdTomato-CENP-A foci. Scale bar = 2.5 µm

Fig. 2

Sp1 regulates chromosome segregation during mitosis. a Schematic describing mAID-Sp1 protein degradation. Created with Biorender.com. b Immunoblot for the indicated proteins in mAID-Sp1 cells in response to 500 µM auxin. Protein lysates were collected at the indicated time points. c Schematic outlining the experimental strategy for (d). Upper panel: mAID-Sp1 cells were arrested in metaphase and collected following the described protocol in (Fig. 1c), spread onto a glass slide, and stained for Sp1 and CENP-A. Scale bars = 10 µm. Squares indicate the inset (lower panel). Scale bars = 1 µm. e Schematic outlining the experimental strategy for (f). f Fluorescent detection of DAPI-stained interphase chromosomes following the indicated treatment. g The percentage of cells harboring micronuclei (white arrows). Scale bar = 10 µm. Minimum 150 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.00056, unpaired t-test. All images are representative

Fig. 3

Sp1 regulates mitotic progression. a and b Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every 3 min, the above image sequence represents images taken every 9 min to best highlight the differences between the treatments. Time = h:min., Scale bar = 5 µm. c Time (m) from nuclear envelope breakdown to G₁. Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.016, unpaired t-test. d Time (m) from nuclear envelope breakdown to anaphase. Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.042, unpaired t-test. e Schematic outlining the experimental strategy for (f). f Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 µm. f Quantification of (e). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.0037, unpaired t-test. All images are representative

Fig. 4

Sp1 regulates mitotic chromosome assembly though condensin complex I localization. a Schematic detailing the experimental strategy for panels b–g. b Left, fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells following the indicated treatment. Scale bar = 10 µm. Squares indicate the inset (right). Scale bar = 1 µm. c Quantification of (b). Minimum 28 cells (1288 estimated chromosomes) counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.012, unpaired t-test. d Left, mAID-Sp1 cells were arrested in metaphase and stained for CAPD-2. Scale bars = 10 µm. Squares indicate the inset (right). Scale bar = 1 µm. e Quantification of (d). Minimum 11 cells (506 estimated chromosomes) counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.015, unpaired t-test. Left, mAID-Sp1 cells were arrested in metaphase and stained for CAP-H2 and SMC4. Scale bars = 10 µm. Squares indicate the inset (right). Scale bar = 1 µm. g Quantification of CAP-H2 intensity in (f). Minimum 19 cells (874 estimated chromosomes) counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.89, unpaired t-test. h Quantification of SMC4 intensity in (f). Minimum 19 cells (874 estimated chromosomes) counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.00098, unpaired t-test. i Representative immunoblot for the indicated proteins in mAID-Sp1 cells in response to 500 µM auxin. Protein lysates were collected at the indicated time points

Fig. 5

Sp1 regulates Aurora B kinase activation early in mitosis. a Schematic detailing the experimental strategy for panels b-e. b mAID-Sp1 cells were arrested in metaphase and stained for p-INCENP. Scale bar = 5 µm. c Quantification of (b). Minimum 29 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.0017, unpaired t-test. d mAID-Sp1 cells were arrested in metaphase and stained for Aurora B kinase. Scale bar = 1 µm. White arrow indicates a misaligned chromosome. e Quantification of (d). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.0035, unpaired t-test. f Left, mAID-Sp1 cells were arrested in metaphase and collected following the described protocol in (Fig. 2c), spread onto a glass slide, and stained for H3^pS10. Scale bar = 10 µm. Squares indicate the inset (right). Scale bar = 1 µm. g Quantification of (f). Minimum 19 cells (874 estimated chromosomes) counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.00018, unpaired t-test. h Representative immunoblot for the indicated proteins in mAID-Sp1 cells following treatment with 500 µM auxin. Protein lysates were collected at the indicated time points. i Quantification of the densitometry normalized to H3 (total) from (g). n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.017, unpaired t-test

Fig. 6

Sp1 regulates mitotic chromosome assembly and segregation during mitosis. Sp1 localizes to mitotic centromeres early in mitosis and regulates Aurora B kinase recruitment, condensin I-mediated chromosome assembly in prophase and ultimately proper segregation of sister chromatids in anaphase. Model created with Biorender.com