ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice
Mathew C. Casimiro, … , Andrew Arnold, Richard G. Pestell
Mathew C. Casimiro, … , Andrew Arnold, Richard G. Pestell
Published February 6, 2012
Citation Information: J Clin Invest. 2012;122(3):833-843. https://doi.org/10.1172/JCI60256.
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Research Article Oncology

ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice

Abstract

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1–/– mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1–rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland–targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.

Authors

Mathew C. Casimiro, Marco Crosariol, Emanuele Loro, Adam Ertel, Zuoren Yu, William Dampier, Elizabeth A. Saria, Alex Papanikolaou, Timothy J. Stanek, Zhiping Li, Chenguang Wang, Paolo Fortina, Sankar Addya, Aydin Tozeren, Erik S. Knudsen, Andrew Arnold, Richard G. Pestell

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Figure 3

Cyclin D1 rescue of Ccnd1–/– MEFs induces polyploidy and aneuploidy.

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Cyclin D1 rescue of Ccnd1–/– MEFs induces polyploidy and aneuploidy. ...
(A) The expression profile for cyclin D1–induced genes (63) was enriched for high CIN score (P < 0.0001). (B) PI staining demonstrated increased polyploidy in cyclin D1–rescued versus control Ccnd1–/– MEFs. (C) Quantitation of PI staining based on 3 separate cell lines (mean ± SEM). *P < 0.005. (D, E, H, and I) Representative metaphases from SKY of control and cyclin D1–rescued Ccnd1–/– MEFs at P6 (D and E) and 3T3 cells at P23 (H and I). Shown for each is an inverted DAPI image of the metaphase (top right), a raw spectral image of the metaphase (top left), and classification of the same metaphase (bottom). (F and J) Chromosomal number across metaphase spreads from control and cyclin D1–rescued Ccnd1–/– MEFs at P6 (F) and 3T3 cells at P23 (J), showing the total number of chromosomes for 20 mitotic spreads. Gray shading represents expected deviation from normal at 2N and 4N (± 2 chromosomes). P < 0.001, rescue vs. control, χ2 test of association. (G and K) Reciprocal translocations and NRTs in metaphase spreads from control and cyclin D1–rescued Ccnd1–/– MEFs at P6 (G) and 3T3 cells at P23 (K), shown as number of events per cell analyzed. The mean distribution is represented as a red curve.