Our present findings indicate that cyclin D1 induces CIN. Cyclin D1 induced gene expression profiles characteristic of CIN in fibroblasts, in the mammary gland, and in cyclin D1–induced mammary tumors. Transient expression of cyclin D1 over 7 days in the mammary gland was sufficient to induce CIN gene expression. SKY analysis confirmed the induction of aneuploidy and polyploidy by cyclin D1 expression in Ccnd1–/–
MEFs. Immunofluorescence demonstrated the occurrence of supernumerary centrosomes that formed multipolar spindles. A careful analysis of the relative abundance of the genes involved in CIN identified a cluster of genes regulating the G2
/M checkpoint and mitosis. The relative abundance of these genes was increased by cyclin D1 expression, as confirmed by quantitative PCR. The finding that cyclin D1 induced CIN is of importance, as CIN is an early feature of tumorigenesis that may precede tumor suppressor loss (39
). Previous studies showed that cyclin E, but not cyclin D1, is capable of inducing CIN (6
). However, cyclin D1 overexpression correlated with aneuploidy, supernumerary centrosomes, and spindle defects in mouse hepatocytes (41
) and with aneuploidy and polyploidy in lymphoid tumors (42
). In addition, cyclin D1 amplification correlated with centrosome amplification in bladder cancer (43
). As cyclin D1 expression is increased in the early phases of tumorigenesis, cyclin D1 may be an important inducer of CIN in tumors.
Analysis of clinical samples with molecular genetic subtyping identified the correlation of the CIN signature with cyclin D1 overexpression and luminal subtype B breast cancer. The presence of CIN in this genetic subtype correlated with poor outcome. Previous studies examining the role of cyclin D1 in outcome have provided contradictory results, with some suggesting a positive correlation between cyclin D1 expression and outcome and others showing reduced survival (16
). Cyclin D1 levels were induced in luminal A and B subtypes, but correlated with CIN in luminal subtype B. CIN is usually poorly tolerated by cells initiating cell death signaling. As luminal A and luminal B breast cancer subtypes have distinct molecular genetic profiles, there may be additional genetic changes in the luminal B tumors that allow the survival of cells with genomic instability. It may well be that the genetic subclassification, as conducted in the current studies, is important in determining the clinical significance of cyclin D1 overexpression. The recent identification of drugs targeting CIN (46
) may provide a rational basis for therapeutic substratification, supplementing with compounds targeting CIN in the luminal B subtype of breast cancer.
Here, we conducted a genome-wide analysis of cyclin D1 binding in the context of local chromatin using ChIP-Seq analysis. Our prior studies demonstrated the recruitment of cyclin D1 in the context of local chromatin to TF binding sites, which was associated with recruitment of SUV39H1 and HP1α and commensurate reduced acetylation and increased trimethylation of H3lys9 (17
). Subsequent studies by ChIP-ChIP covering –5.5 to +2.5 kb of a subset of promoters similarly identified cyclin D1 recruitment to a subset of target genes involved primarily in notch signaling and cellular proliferation (28
How might cyclin D1 regulate gene expression in the context of local chromatin? Although intrinsic DNA sequence–specific binding of cyclin D1 has not been identified, cyclin D1 has been identified at sites of damaged DNA in the context of local chromatin (48
). Various TFs associate with cyclin D1 in IP–Western blot analysis, and the abundance of cyclin D1 can regulate the recruitment of TFs (22
) and transcriptional coregulators (26
) in the context of local chromatin in ChIP assays. Given these findings, we had proposed that cyclin D1 is recruited either to DNA through sequence-specific binding proteins to regulate gene expression or to damaged DNA via Rad51 and the related repair complex, which thereby recruits BRCA proteins (29
). Cyclin D1 abundance determines the recruitment of cointegrator and chromatin remodeling proteins in ChIP assays, including p300/CBP (26
), SUV39H1, HP1α, and HDAC1/3 (17
), and dictates acetylation and dimethylation of local histones (e.g., H3 and H4). The mechanisms permitting assembly of the cointegrator regulatory complex that are associated with cyclin D1 at a given cis
element remain to be determined. Prior studies using cyclin D1 and p300 knockout mice showed that, in the case of genes governing the fidelity of DNA replication (e.g., MCM3, MCM4, and RfCH), their abundance was induced by cyclin D1 and reciprocally regulated by p300, consistent with previous findings that cyclin D1 inhibits p300 autoacetylation (26
). Although the regulation of TFs and cointegrator activity was independent of the cdk-binding domain, the role of the cyclin D1 cdk-binding domain in regulating the CIN signature in vivo remains to be determined.
The current studies identified a distinct subset of cis
elements occupied by cyclin D1, due in part to the distinct interrogation of the genome conducted herein. The current studies examined both noncoding and coding DNA and sites distal to the transcription start site and identified a proclivity for cyclin D1 to occupy the CTCF binding factor site. CTCF functions in chromatin reorganization and as an enhancer insulator (50
). It is of interest that the cohesin complex — important in segregation of sister chromatids, which were altered in a cyclin D1–dependent manner — interacts with CTCF. The cohesin complexes are also found at a large fraction of CTCF sites in vivo (51
). Because CTCF is a chromatin reorganizer and has the potential to play a bidirectional role through the cohesin complex, it will be of interest to determine the relative importance of cyclin D1 in regulating CTCF-dependent global transcription.