BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin

Abstract

Breast cancer-associated protein 1 (BRCA1) forms foci at sites of induced DNA damage, but any significance of these normal S-phase foci is unknown. BRCA1 distribution does not simply mirror or overlap that of replicating DNA; however, BRCA1 foci frequently abut sites of BrdU incorporation, mostly at mid-to-late S phase. Although BRCA1 does not overlap XIST RNA across the inactive X chromosome, BRCA1 foci position overwhelmingly in heterochromatic regions, particularly the nucleolar periphery where many centromeres reside. In humans and mice, including early embryonic cells, BRCA1 commonly associates with interphase centromere-kinetochore complexes, including pericentric heterochromatin. Proliferating cell nuclear antigen or BrdU labeling demonstrates that BRCA1 localizes adjacent to, or "paints," major satellite blocks as chromocenters replicate, where topoisomerase is also enriched. BRCA1 loss is often associated with proliferative defects, including postmitotic bridges enriched with satellite DNA. These findings implicate BRCA1 in replication-linked maintenance of centric/pericentric heterochromatin and suggest a novel means whereby BRCA1 loss may contribute to genomic instability and cancer.

Figure 1.

BRCA1 and XIST RNA/Xi relationship in human female fibroblasts. (A and B, TIG1; C, WI38; D, IMR90). (A) An optical section from a deconvolved stack, followed by a line scan of the fluorescence intensities. At right, 3D rendering with two views rotated ∼20 degrees is shown (Video 1). (B) Unprocessed micrograph followed by a magnified view of a deconvolved optical slice showing XIST RNA (red) and BRCA1 (green). (C) BRCA1 (green) and a replicating Barr body (BrdU, red; DAPI, blue). (D) X centromere (red) and BRCA1 (green) with DAPI (gray).The DAPI-dense Barr differentiates Xa from Xi. Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.200602055.

Figure 2.

BRCA1 relationship to the heterochromatic compartment and replication in human fibroblasts. (A) BRCA1 (red) and Cot1 hybridization to hnRNA (green; DAPI, blue). (B) BRCA1 (green) and SC-35 domains (red). (C) BRCA1 (green) relative to BrdU-labeled, late-replicating DNA (red). (D and E) BRCA1 association increases during replication. (D) The replicating Xi in human cells was distinguished by BrdU and DAPI staining for the Barr body (n = 100). (E) Replicating mouse chromocenters were distinguished by PCNA label (n = 1100).

Figure 3.

BRCA1 and human centromere markers in interphase fibroblasts. (A) BRCA1 (red) and CENP-C (green) in TIG1 cells. (B) BRCA1 (green) and centromeric DNA (red) in IMR90. (C) BRCA1 (green) and CENP-B (red) in TIG1. Asterisks mark sites of association highlighted in insets and arrowheads mark some of the other sites of association.

Figure 4.

BRCA1, mouse chromocenters, and the relationship to replication. (A) Mouse cell lines included MEFs (BRCA1, green), 3T3 (BRCA1, red), and mouse ES cells (BRCA1, green). (B) BRCA1 (red) and minor satellite DNA (green). Far right shows 3D-rendered image of BRCA1 and minor satellite on a chromocenter (blue). Middle photo shows chromocenters in black. (C) Cells were labeled for replication with PCNA (red) and BRCA1 (green). In top panel, the upper nucleus has more BRCA1 and PCNA on chromocenters. (D and E) topoII-α (green) on mouse chromocenters (blue) is seen in many S-phase cells (PCNA, red).

Figure 5.

Centromeric markers and DNA bridges in BRCA1−/− HCC1937 cells. (A) TIG1 (left) and HCC1937 cells (right) stained for CENP-A and -F. (B) In HCC1937 early G1 daughter cells, DAPI staining shows thin DNA bridges. (C) Centromeric DNA (green) in DNA bridges (left and right) and lagging chromosomes (middle) in HCC1937 cells.