BRCA1 tumour suppression occurs via heterochromatin-mediated silencing

Abstract

Mutations in the tumour suppressor gene BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of Brca1 in mice results in transcriptional de-repression of the tandemly repeated satellite DNA. Brca1 deficiency is accompanied by a reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions and ubiquitylates H2A in vivo. Ectopic expression of H2A fused to ubiquitin reverses the effects of BRCA1 loss, indicating that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA de-repression was also observed in mouse and human BRCA1-deficient breast cancers. Ectopic expression of satellite DNA can phenocopy BRCA1 loss in centrosome amplification, cell-cycle checkpoint defects, DNA damage and genomic instability. We propose that the role of BRCA1 in maintaining global heterochromatin integrity accounts for many of its tumour suppressor functions.

Figure 1. BRCA1 deficiency impairs heterochromatin structure

a, Expression of BRCA1 is diminished in the Nestin-Cre BRCA1 knockout brains as shown by immunoblotting. KO: samples from a P7 BRCA1 KO (Brca11+/-;Brca5-13cK+/-;nestin-Cre+) mouse. CTRL: samples from a control P7 (Brca11+/-; Brca5-13cK+/-; nestin-Cre-). BRCA1 immunoblot (upper panel), tubulin loading control (lower panel). b, Lack of BRCA1 induces changes in the nuclear morphology of P7 KO cortical cells. The numbers of strong DAPI staining nuclear foci/cell were counted and their frequencies plotted (right panel). c, Confocal microscopic images of brain sections from BRCA1 conditional KO and control mice (P7) stained with antibodies against HP1 and ubiquitin-H2A (UbH2A).

Figure 2. BRCA1 and its ubiquitin E3 ligase activity are required for gene silencing in constitutive heterochromatin

a, Quantitative RT-PCR showed that heterochromatic regions or certain imprinted genes (Igf2 & H19 but not Igf2R) were upregulated in BRCA1 KO brains. Internal controls: cyclophilin or 18SRNA. b, Rescue of the BRCA1 mediated repression in NPCs by exogenous human BRCA1 but not GFP after infection with retroviral CRE-GFP. c, Quantitative RT-PCR experiments showed that heterochromatic regions or imprinted genes were upregulated in BRCA1 KO mammary glands (6-week virgin). d, The ubiquitin E3 ligase activity of BRCA1 is required for heterochromatin silencing. HCC1937 cells were reconstituted with either wild type BRCA1 (HCCB1) or BRCA1 with a mutation in the RING domain, T37R. P7 cerebellar ChIP experiments with antibodies against (e) mouse BRCA1 or VSVG protein as a control and (f) ubiquityl-histone H2A or histone H3 protein as a control. Enrichment of major and minor satellite DNA was measured by quantitative PCR relative to 18S control. g, ChIP experiments were performed using ubiquitinated histone H2A antibody as in (f) and HCC1937 cells reconstituted with GFP, BRCA1 (V11A) or a mutant (T37R). Error bars are shown as s.d. Each result shown is representative of three independent experiments.

Figure 3. Ubiquitinated histone H2A is directly involved in BRCA1 mediated heterochromatic silencing

a, A diagram showing the artificial ubiquitin-H2A expression vector. b, HCC1937 cells were infected with a lentiviral vector expressing ubiquitylated histone H2A mimic fusion protein or GFP as a control and the levels of alpha satellite variants (mcBox, SATa and SATIII) transcription were measured by qRT-PCR. c, Ectopic expression of ubiquitylated histone H2A re-established heterochromatin silencing in BRCA1 null NPCs. NPCs were infected with GFP alone, ubH2A, CRE-GFP alone and CRE-GFP coinfected with UbH2A. The levels of satellite DNA transcription were measured by qRT-PCR. d, NPCs showed increased proliferation upon expression of ubiquitylated histone H2A in BRCA1 null NPCs. Two days after infection (2 dpi), cells were labeled with BrdU prior to fixation, immunostaining and FACS analysis. Result shown is representative of 3 independent experiments. e, NPCs showed reduced apoptosis upon expression of ubiquitylated histone H2A in BRCA1 null NPCs. NPCs were cultured and infected as in (d). Cells were fixed, immunostained with anti-activated caspase 3 antibody and subjected to confocal imaging. Error bars are shown as s.d. Scale bar: 100 μm

Figure 4. Derepression of satellite DNA transcription occurs in BRCA1-deficient breast cancers

a, Haematoxylin/eosin staining of BRCA1KO mouse breast cancer. Because BRCA1 KO female mice develop mammary tumors very rarely, these mice were crossed into a p53 heterozygous background for accelerated tumorigenesis. MG, mammary gland from normal mouse littermates. MGT, mammary tumors from BRCA1KO mouse (Brca11+/-;Brca5-13cK+/-;MMTV-Cre+;p53+/-) developed at 6 months. b, Quantitative RT-PCR experiments showed that heterochromatic regions and imprinted genes were upregulated in mouse BRCA1 KO breast cancer in comparison to that of wildtype mammary glands from littermates (internal control 18SRNA). The result shown is an aggregate of 5 mouse independent sample pairs. c, Quantitative RT-PCR experiments showed the satellite DNA transcripts, CFXr and SATa, significantly derepressed (Student’s 2-tailed t-test) in human BRCA1-mutant breast tumors (n=8) in comparison with that of normal breast tissues (n=11) . Ct values of each sample were normalized with GAPDH. Error bars are shown as s.d.

Figure 5. Ectopic expression of satellite DNA leads to genomic instability in human mammary epithelial cells

a, A diagram of the lenti-viral vector expressing the human or mouse satellite DNA sequence (red arrows) under the H1 promoter (gray arrow). b, c, d, Overexpression of satellite RNA induced mitotic catastrophe (b), centrosome amplification (c), histone γH2Ax phosphorylation (d), in primary HMECs. e, Defective mitotic checkpoints induced by satellite DNA overexpression. 48h after transduction of a lentivirus expressing human satellite DNA sequence with forward direction (human satellite F), or with reverse direction (human satellite R), or mouse satellite DNA sequence, or an empty vector, U-2OS cells were blocked with thymidine, followed by treatment of nocodazole. The cells were stained with phosho-histone H3 antibody and PI prior to flow cytometric analysis. The shown experiment is representative of 3 independent replicates.