Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen

Abstract

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein.

Figure 1. Proteomic analyses of cyclin D1-associated proteins

a , Silver-stained gels with cyclin D1-containing complexes purified from indicated organs (D1-Ip). Mock-Ip, mock purification from organs of wild-type mice. Cyclin D1 is marked by an asterisk. b, Relative abundance (arbitrary units, au) of Cdks and cell cycle inhibitors in cyclin D1-containing complexes in different compartments. c, Fraction of proteins among high-confidence cyclin D1-intactors and among “mock” purified proteins classified to indicated gene ontology categories.

Figure 2. Analyses of cyclin D1 interaction with the mouse genome

a, Scatterplot of chromatin immunoprecipitation (Ip) with anti-FLAG antibody. Log₂ intensity values of Ip from knock-in embryos are plotted against values from wild-type embryos. b, Examples of cyclin D1-bound regions. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. c, Location of cyclin D1 binding sites relative to transcription start sites of RefSeq genes. d, Conserved DNA sequence motifs identified among cyclin D1-bound regions. e, Left panel: Unsupervised clustering of cyclin D1-binding events, identified in whole-embryo ChIP-chip. Each horizontal line represents one gene, centered around transcriptional start site. Yellow and blue depict bound and unbound probes. Right panel: Number of tags observed for a given transcript (yellow: ≥4 tags; blue: ≤3 tags).

Figure 3. Analyses of cyclin D1 transcriptional function in retina

a, Expression pattern of genes whose promoter regions were bound by cyclin D1, and which showed altered levels in D1^−/− retinas. b, Binding of cyclin D1 to regulatory regions of indicated genes verified by targeted ChIP with anti-FLAG antibodies (to bring down cyclin D1) using postnatal day 0 retinas. c, d, Expression levels of indicated genes quantified in wild-type and D1^−/− retinas by RT-PCR. Shown is fold-difference between wild-type versus D1^−/− retinas. e, Levels of Notch1 protein in retinas, determined by immunoblotting. f, Levels of Notch1 transcripts in R28 cells, quantified by RT-PCR following knock-down (+shCycD) or overexpression of cyclin D1 (+CycD1). Shown is fold-difference compared to cells infected with control vector. Error bars, SD.

Figure 4. In vivo and molecular analyses of cyclin D1 – Notch1 connection

a, Whole mounts of D1^−/− retinas infected with viruses encoding activated Notch and β-galactosidase (NIN-NICD, see Supplemental Fig. 10) or with vectors encoding only β-galactosidase (NIN), stained with X-gal to visualize clones of infected cells (arrowheads). b, higher magnifications of a. c, percentage of β-galactosidase-positive clones composed of 1, 2, 3 and ≥4 cells. d, Cyclin D1 was immunoprecipitated from P0 retinas and probed with indicated antibodies. e, Targeted ChIP on P0 retinas using anti-FLAG (to bring down cyclin D1) or with anti-CBP antibodies, followed by real-time PCR with Notch1-specific primers. Lower panel: ChIP with anti-FLAG followed by re-ChIP with anti-CBP antibodies and real-time PCR. f, Cyclin D1 was knocked-down (+shCycD1) or overexpressed (+CycD1) in R28 cells. ChIP with anti-cyclin D1, anti-CBP, anti-acetylated histone H4K5 (AcH4K5) or anti-acetylated histone H3K9,14 (AcH3K9,14) antibodies was followed by real-time PCR with Notch1-specific primers. The results show fold-difference compared to cells transduced with empty vectors. g, ChIP using anti-CBP and anti-AcH4K5 antibodies followed by real-time PCR with Notch1-specific primers using wild-type and D1^−/− retinas. Error bars, SD.