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, T37R²³. 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.