Dynamic long-range association between regulatory regions of BRCA1. (A) Diagram of BRCA1, exons 1–24 (bold lines) flanked by 3 kb upstream of exon 1 and 2 kb downstream of exon 24 (thin lines) with BanI (above sequence) and DpnII (below sequence) sites indicated. Upright tall arrows indicate divergent transcription initiation positions for NBR2 and BRCA1. BRCA1 exons are indicated by open rectangles, and an NBR2 exon is indicated by a striped rectangle. The gray rectangles indicate regions known to possess regulatory (promoter, enhancer, or repressor) activity. Vertical lines represent restriction sites. Black labeled arrows indicate primer direction. Checked boxes represent restriction fragments involved in gene looping based on 3C analysis. (B) (i) BanI 3C analysis of BRCA1 in the MCF7 breast adenocarcinoma cell line before and after 5 or 24 h of 100-nM estrogen stimulation and artificial 3C products (see Materials and Methods) using primer B2 in combination with one of the other BanI primer as indicated. BRCA1 load represents the product of PCR analysis of the same BanI 3C using BRCA1-specific internal primers in intron 2. (ii) qRT-PCR analysis of BRCA1 mRNA, relative to 18S rRNA, after induction with 100 nM estrogen showing a slight increase after 5 h and a significant increase after 24 h. (iii) qRT-PCR analysis of BRCA1 mRNA and pre-mRNA, relative to 18S rRNA, after induction with 100 nM estrogen showing a significant increase in both after 24 h of stimulation. (iv) Quantitation of 3C band intensity relative to intensity of 3C control bands (see Materials and Methods). (C) 3C and Western analysis of MCF7 cells that are either unstimulated or stimulated with 100 nM E2 for 24 h and then untreated or treated with the transcription inhibitor KM05283 for 1 h. (i) 3C analysis of MCF7 cells grown in serum-free phenol red free media (−e) or stimulated with 100 nM estrodiol for 24 h (+e), and either untreated (−K) or treated with KM05283 for 1 h (+K). 3C-positive controls and BRCA1 loading control were as for Bi. (ii) Quantitation of 3C band intensity relative to intensity of 3C control bands (see Materials and Methods). (iii) Western analysis with antibodies against Pol II Ser-2 phosphoylated CTD (Ser 2P) or the N terminus of the large subunit of Pol II showing that the transcription inhibitor has worked. Anti-Oct antibody provides loading control.

Repression of mouse Brca1 is also associated with long-range associations. (A) qRT-PCR of Brca1 mRNA levels relative to 18S rRNA in mammary glands from mice at day 15 of pregnancy (Preg #1) or day 9 of lactation (Lact #1 and Lact #2). Quantitation was performed by using the comparative ΔCt method as described in Materials and Methods. (B) BamHI 3C analysis of Brca1 in pregnant and lactating mammary glands. (i) Map of mouse Brca1 showing BamHI restriction sites and relevant exons and primers as above. (ii) 3C analysis of chromatin extracted from pregnant and lactating mammary glands from two independent mice (nos. 1 and 2). 3C-positive controls and Brca1 and MyoG loading controls are as described above and in Materials and Methods. (iii) Quantitation of 3C band intensity relative to intensity of 3C control bands (see Materials and Methods).

Estrogen-responsive promoter-terminator gene loop of BRCA1. (A) SacI 3C analysis across BRCA1, before and after estrogen stimulation. (i) Map of BRCA1 showing SacI restriction sites with relevant exons and primers. 3C-positive fragments are indicated by checked boxes. (ii) SacI 3C analysis of MCF7 cells grown in serum-free phenol-red free media (0 h) or treated with 100 nM E2 for 5 or 24 h. 3C-positive controls and loading controls are as for Fig. 1. (iii) SacI 3C analysis of three different human breast cancer cell lines grown in complete media (T47-D, ZR-75, MDA-MB-468) show differential chromatin loops. (B) (i) Map showing the overlap between interacting 3C fragments identified by using three different restriction fragments and the corresponding sequences used to examine the regulatory activity of BRCA1 promoter and terminator sequences in reporter assays (dark shaded boxes). (ii) Luciferase activity in cells transfected with reporter constructs containing combinations of the SV40 or BRCA1 promoter and the SV40 and BRCA1 terminator region and either grown in serum-free phenol-red free media (empty bars) or stimulated with 100 nM E2 (filled bars). Data shows that the significant induction of the BRCA1 promoter by E2 (P = 0.008) is suppressed in the presence of the BRCA1 terminator (P = 0.67).

Proposed model of BRCA1 gene looping. Diagram shows two possible states of the human BRCA1 gene during transcriptional regulation. (Left) A factor (black) mediates a long-range association between the BRCA1 promoter and terminator, which in turn drives formation of a chromatin loop preventing further transcription. We refer to this as regulated repression of BRCA1, based on our finding that loop formation is transcription-dependent. (Right) When transcription of BRCA1 is induced (e.g., upon estrogen stimulation), we propose that expression of another factor (red) is induced that interferes with the function of the black factor, causing chromatin to become relaxed and promote transcription.