Is there more to BARD1 than BRCA1?

Biology of Aging Laboratory, Department of Geriatrics and Department of Gynecology and Obstetrics, Geneva University and University Hospitals, 30, Bloulevard de la Cluse, CH-1211 Geneva, Switzerland. Correspondence to I.I.-F. e-mail: [email protected]:10.1038/nrc1878

RING-finger domainThe RING-finger domain, first identified in the RING (really interesting new gene) protein, describes a specific cysteine-rich class of Zn2+-binding Zinc-finger motifs. RING-finger domains are found as DNA-binding domains and as protein-interaction domains. Recently, ubiquitin ligase activity has been attributed to many RING-finger proteins.

Is there more to BARD1 than BRCA1?Irmgard Irminger-Finger and Charles Edward Jefford

Abstract | It has been over a decade since mutations in BRCA1 and BRCA2 were found to be associated with a small number of familial breast cancer cases. BRCA1 is a large protein that interacts with many other proteins that have diverse functions, so it has been a challenge to determine how defects in its function could lead to cancer. One particular protein, BARD1, seems to be an important regulator of the tumour-suppressor function of BRCA1, as well as acting as a tumour suppressor itself. BARD1 is indispensable for cell viability, so loss-of-function mutations are rare, but mutations and truncations that alter its function might be involved in the pathogenesis of breast cancer.

About 10% of women diagnosed with breast cancer have inherited mutations in BRCA1 (REF. 1) or BRCA2 (REF. 2) that have been associated with predisposition to breast and ovarian cancer. The BRCA1-associated ring domain 1 (BARD1) protein was discovered in a yeast two-hybrid screen as a binding partner of BRCA1 (REF. 3). BARD1 and BRCA1 have several fea-tures in common: similar protein structure (FIG. 1), the embryonic lethality of their respective knockout mice and induction of genetic instability when depleted from cells.

BARD1 and BRCA1 form a functional heterodimer through the binding of their RING-finger domains3,4. This interaction is thought to stabilize both proteins, as the respective monomers are unstable5,6. Furthermore, the BRCA1–BARD1 interaction is required for several of the cellular and tumour-suppressor functions of BRCA1.

Few cancer-associated mutations have been found in BARD1, compared with more than 650 (Human Gene Mutation Database) for BRCA1. These findings have been interpreted to mean that BARD1 is only an accessory protein for BRCA1. However, several reports have demonstrated BRCA1-independent functions of BARD1, primarily in apoptosis, and BRCA1-independent increased expression of BARD1 during mitosis, indicating that BARD1 on its own might have crucial functions.

BARD1 — a unique proteinThe similarities between BARD1 and BRCA1 are evi-dent (FIG. 1): both proteins have a RING domain (which mediates DNA–protein and protein–protein inter-actions), a nuclear export signal at their N termini and two tandem BRCA1 carboxy-terminal (BRCT) domains. BARD1 and BRCA1 have no sequence or structural similarity with BRCA2. The interaction of BRCA1 and

BARD1 is required for tumour suppression, as specific mutations within the BRCA1 RING domain are associ-ated with breast and ovarian tumours3.

Although many functions of the BRCA1–BARD1 heterodimer depend on the RING domains and adja-cent regions, the function of the BRCT domains, which are also protein–protein interaction domains, is less well understood. A potential function of the BRCT domain of BRCA1 is in binding to the substrates of the DNA damage response kinases, such as ataxia tel-angiectasia mutated (ATM)7,8. The biological relevance of this interaction is supported by tumour-associated mutations in the BRCT domains of BRCA1 that result in the loss of phospho-epitope binding activity. The function of the BARD1 BRCT domains, and whether or not they contribute to the function of the BRCA1 BRCT domains, has not been investigated. In addition to RING and BRCT domains, BARD1 has three ankyrin repeats (ANK) that also facilitate protein–protein inter-actions. Interestingly, no other proteins that contain RING, ANK and BRCT domains were identified in a database search using the Simple Modular Architecture Research Tool (SMART).

BARD1 orthologues have been identified in mouse, rat, Xenopus laevis6, Caenorhabditis elegans9, and Arabidopsis thaliana10. The domain structure and the intron–exon boundaries that lie within the regions that encode the RING domain, and the region that includes the ANK repeats through to the BRCT domains, are conserved between the species. In addition to RING, ANK and BRCT domains, protein-sequence analysis predicts that BARD1 might possess an uncharacter-ized domain (I.I.-F., unpublished observations) that is located between the ANK and BRCT domains, which is also highly conserved in various species (FIG. 1). This complexity of structure indicates that BARD1 could

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Mouse BARD1 765 aa

777 aa

1823 aa

Human BARD1

BARD1

Human BRCA1

66 95 53 97 77 91 Percentage of identity

RING

NES

NES

NLS BRCT

a

b c

P24S S241C

R378SR358D

K153E

L312NN295S

Q406RN470S

V507M

C557SR658C

Q564H

V695L

S761N

BARD1βTestes

BARD1γTestes

BARD1δOvarian cancer cellsHeLa cells

In1 In2 In3 In4In5 In7 In8 In9

In10In6

P P PP

Loss of heterozygosity(LOH). In cells that carry a mutated allele of a tumour-suppressor gene, the gene becomes fully inactivated when the cell loses a large part of the chromosome carrying the wild-type allele. Regions with a high frequency of LOH are believed to harbour tumour-suppressor genes.

Clear cell adenocarcinomaLike most ovarian cancers, the clear cell carcinoma has a surface epithelial origin. Clear cell carcinomas are characterized by large epithelial cells with abundant clear cytoplasm. Clear cell carcinomas are dedifferentiated, highly aggressive tumours, and the 5-year survival rate is less than 50%.

have multiple functions. These might be regulated by the expression of differentially spliced isoforms and post-translational modifications.

BARD1 mutations and cancerBecause mutations in BRCA1 and BRCA2 account for only 50% of familial breast and ovarian cancer cases (see data in the Human Gene Mutation Database), BARD1 mutations were expected to account for additional cases of inherited and sporadic breast and ovarian cancers. Screening of patients with sporadic breast, ovarian and endometrial cancers identified three missense alterations at amino-acid positions Q564H, V695L and S761N, and

loss of heterozygosity (LOH) was associated with Q564H and S671N mutations11 (FIG. 1b). The Q564H mutation was also found in the germ line of a patient with clear cell adenocarcinoma11. In a screen of an Italian cohort of familial breast and ovarian cancers that were not associated with BRCA1 and BRCA2 gene mutations, five alterations in BARD1 were discovered12, including 1139del21 and C557S, which was previously described as a polymorphism11. The C557S mutation was also found to be associated with hereditary susceptibility to breast cancer in a Finnish population study13. Analysis of BARD1 in Japanese patients with familial breast cancers who did not carry BRCA1 or BRCA2 germline mutations revealed six alterations, including the 1139del21 muta-tion (previously identified in the Italian cohort12) and N470S, which was identified as a germline mutation14. In a Geneva study of ovarian cancers, N-terminal deletions in BARD1 were discovered in 70% of the cDNAs ana-lysed. The remaining 30% carried a Q406R mutation15.

So, BARD1 mutations are associated with a few cases of spontaneous breast and ovarian tumours that are not caused by BRCA1 or BRCA2 mutations, and account for only a small fraction of cases of familial breast cancer overall. This could mean that BARD1 mutations are not important features in the develop-ment of gynaecological cancers, or that BARD1 defi-ciency is deleterious to the cell. The latter hypothesis is supported by the finding that homozygous disruption of Bard1 in mice results in early stage lethality in utero (embryonic day 7.5), owing to proliferation defects and a high level of genomic instability16. As BRCA1

At a glance

• BRCA1-associated ring domain 1 (BARD1) is the main binding partner of BRCA1 and is essential for the tumour-suppressor functions of BRCA1.

• The BARD1–BRCA1 heterodimer has ubiquitin ligase activity that targets proteins involved in cell-cycle regulation and DNA repair for degradation.

• BARD1 has a BRCA1-independent function in mediating p53-dependent apoptosis. It binds to p53, facilitating its phosphorylation and stabilization.

• BARD1 is expressed in most proliferative tissues, including that of the breast, ovary and uterus. It is transcriptionally upregulated in response to DNA damage, hypoxia and hormone signalling. Its translation is activated by cell-cycle-dependent phosphorylation.

• BARD1 is mutated or truncated in breast, ovarian and uterine tumour samples.

• Depletion of BARD1 leads to early embryonic lethality in mice, and genomic instability in vitro and in vivo; phenotypes that are also observed for BRCA1 or BRCA2 deficiencies.

Figure 1 | BARD1 protein. a | The BRCA1-associated ring domain 1 (BARD1) domain structure is compared to that of BRCA1. RING (green), ankyrin (ANK, blue), BRCA1 carboxy-terminal (BRCT, red) domains, and location of potential nuclear localization signal (NLS, light blue) and nuclear export signals (NES, brown) are indicated. Evolutionary conservation is indicated as the percentage of identical amino acids between the mouse and human sequences within distinct regions. b | Phosphorylation and mutation sites of the human BARD1 protein. Phosphorylation sites are marked with P. Mutations are marked in red, germline mutations in blue, and polymorphisms in black. RING (green), ANK (blue), and BRCT (red) motifs are indicated. c | BARD1 splice variants. BARD1β and γ are expressed by preleptotene spermatocytes59, whereas BARD1δ is overexpressed in an ovarian cancer cell line17 and in HeLa cells18. Note that the N-terminal exons 2–6 are frequently lost in ovarian cancer samples15. In, intron.

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BARD1 777 aaP PPP

Homodimer

Ubiquitin ligase function

EssentialNLS

F1 immunogenicregion

Phosphobindingregion

BRCA1

Nucleophosmin/B23

p53

CSTF1

NF-κB (BCL3 and IκBα) ANK repeats

EWS and EWS/FLI1

Minimal apoptoticregion

*

Ubiquitin ligase (E3)Proteins targeted for degradation by the proteasome are selected by having covalently linked ubiquitin chains. This ubiquitylation requires a ubiquitin-conjugating enzyme and a ubiquitin-protein ligase. The importance of E3s is highlighted by the number of normal cellular processes they regulate and the number of diseases that are associated with their loss of function or inappropriate targeting.

levels are also decreased in the Bard1–/– embryos (as are BARD1 levels in Brca1–/– embryos16) a contribution of BRCA1 to BARD1-dependent lethality, and vice versa, cannot be excluded.

Surprisingly, immunohistochemical analyses showed that BARD1 was overexpressed in tumours with BARD1 mutations, as well as in tumours with familial BRCA1 mutations and in a series of sponta-neous ovarian, breast and lung cancers. Increased expression levels of BARD1 were seen exclusively in the cytoplasm, whereas a low level of expression was found in the nuclei of cells in adjacent healthy tissue15. Indeed, in ovarian clear cell adenocarcinoma 100% of cancer cells expressed cytoplasmic BARD1. Overall, upregulated BARD1 expression in breast and ovarian cancers is positively correlated with poor prognostic indicators, such as ovarian tumour type or breast tumour size and stage.

In most cases of ovarian cancer, the N-terminal epitopes of BARD1 were deleted, indicating that tumorigenesis might involve only specific forms of BARD1. These isoforms could have lost tumour-suppressor functions and possibly acquired onco-genic potential. In support of this view, a rat ovarian cancer cell line that lacks full-length BARD1, but expresses a splice variant that is missing exons 2–6 (BARD1δ), is resistant to apoptosis (FIG. 1b). Apoptosis could be induced in these cells by expression of full-length BARD1 (REF. 17). The BARD1δ isoform is also expressed in HeLa cells, in addition to full-length BARD1 (REF. 18). More work from larger cohorts is awaited to corroborate these data and to establish whether the expression of aberrant forms of BARD1 will be effective prognostic markers.

Consistent with the concept of an oncogenic form of BARD1 that is expressed in cancer cells, an increased expression level of BARD1 was described as one of the markers for treatment failure in patients with CNS embryonal tumours19. Furthermore, BARD1 has been shown to interact with the Ewing sarcoma gene product (EWS) and the oncogenic fusion protein EWS–FLI1 (REF. 20) (FIG. 2), which is consistent with an oncogenic function of BARD1. Alternatively, EWS might seques-ter BARD1, therefore inhibiting its tumour-suppressor functions.

Functions of the BRCA1–BARD1 heterodimerUbiquitin ligase activity. A breakthrough in under-standing the mechanism of BRCA1–BARD1 function came from the finding that this heterodimer has ubiqui-tin ligase activity21–24. Individually, BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro. It is possible that this is due to the decreased protein stabil-ity of the monomers. Several groups have established that the cancer predisposing mutations C61G and C64G, which lie within the RING domain of BRCA1, abolish the E3 ubiquitin-ligase activity and tumour-suppressor function of BRCA1–BARD1 (REFS 4,21,25) but do not disrupt the BRCA1–BARD1 inter-action in vitro4,26, despite the fact that these residues were originally reported as being crucial for the inter-action of BRCA1 with BARD1 in a yeast two-hybrid assay3. Cells that express these mutant forms of BRCA1 are also hypersensitive to γ-irradiation, indicating that the BRCA1–BARD1 ubiquitin ligase function is linked to DNA repair pathways25.

In theory, polyubiquitylation can occur by isopep-tide bond formation that involves any of the seven lysine residues of ubiquitin. Isopeptide linkage of ubiquitin using K48 commonly targets proteins for degradation by the proteasome. The BRCA1–BARD1 heterodimer directs polymerization of ubiquitin primarily through K6, an unconventional linkage27. BRCA1 and BARD1 also undergo auto-ubiquityla-tion at non-K48 ubiquitin residues, which increases the ubiquitin ligase activity of the heterodimer by 20-fold27–29. BRCA1–BARD1 auto-ubiquitylation and K6-linked conjugated ubiquitin structures are observed at sites of DNA replication and repair23. This indicates that auto-ubiquitylation of BRCA1 and BARD1 might regulate BRCA1– BARD1 ubiquitin ligase activity and induce DNA repair pathways.

BRCA1–BARD1 targets. BRCA1 localizes to the centro-some during mitosis30. Centrosome amplification, defec-tive G2–M checkpoint control and genetic instability are found in cells from mice with a targeted deletion of exon 11 of BRCA1 (REF. 31). The centrosome component γ-tubulin is ubiquitylated by BRCA1–BARD1 in vitro, using both K48 and K344 residues32. Expression of a γ-tubulin K48R mutant protein caused a marked amplification of the centrosomes. This result links the BRCA1–BARD1 ubiquitin ligase function to cell-cycle checkpoint functions. This particular function of BRCA1 might be regulated at the G2–M checkpoint through

Figure 2 | Protein interactions and functional domains of BARD1. RING (green), ankyrin (ANK, blue), BRCA1 carboxy-terminal (BRCT, red) domains, and location of potential nuclear localization signals (NLS, light blue) are indicated. P indicates the position of phosphorylated serines or threonines, P* indicates phosphorylation that competes for BRCA1 binding and ubiquitin ligase functions. Regions required for homodimer formation and ubiquitin ligase activity are labelled at the N terminus. The minimal apoptotic region, F1 immunogenic regions68 and phosphobinding regions are marked at the C terminus. Interacting proteins are shown as bars covering the approximate region of interaction with the BRCA1-associated ring domain 1 (BARD1). CSTF1, cleavage stimulation factor subunit 1 50kDa; EWS, Ewing sarcoma gene product; IκBa, NF-κB inhibitor, alpha.

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Box 1 | NPM structure and function

Nucleophosmin (NPM) is mutated in human tumours and is also the site of chromosomal translocations that are associated with haematological disorders. Deletion of Npm in mice produces a phenotype similar to that of Brca1 — or BRCA1-associated ring domain 1 (Bard1)-null mice — embryonic lethality after day 11, genomic instability associated with unrestricted centrosome duplication and, in heterozygous mice, a predisposition to the development of haematopoietic disorders35. NPM also has anti-apoptotic functions that are thought to depend on NPM binding to various tumour-suppressor proteins86. The diagram shows the overlapping functions of NPM, BRCA1–BARD1 and BARD1. The BRCA1–BARD1 heterodimer has been shown to ubiquitylate (Ub) NPM. In addition, BRCA1–BARD1 regulates centrosome number, and increased expression levels of NPM lead to centrosome amplification. Another mechanistic overlap between these proteins occurs with the CDK2–cyclin E complex. The BRCA1–BARD1 ubiquitin ligase activity is thought to be downregulated by the CDK2–cyclin E complex, whereas NPM is phosphorylated (P) and stabilized by CDK2–cyclin E. NPM can also sequester the tumour suppressor ARF, influencing p53-mediated cell-cycle arrest and apoptosis.

Ub

P

Ub

P

UbiquitinE3 ligase

BRCA1

BARD1

CDK2

Cyclin E

Proteasome degradation

γ-Tubulin

Ub

NPM

ARF

Cell-cycle progression

Centrosome amplification

p53 expression

Cell-cycle arrest and apoptosis

Nuclear dotsThe term nuclear dots describes the local accumulation of proteins within the nucleus, as detected by immunostaining.

phosphorylation by Aurora A, a kinase that also localizes to the centrosome and regulates the G2–M transition33. It seems logical that the BRCA1–BARD1 mediated ubiquitylation of γ-tubulin could depend on BRCA1 phosphorylation.

BRCA1–BARD1 also ubiquitylates the nucleolar phosphoprotein nucleophosmin (NPM, also known as B23). NPM interacts with BRCA1– BARD1 and co-localizes with BARD1 and BRCA1 in mitotic cells. Exogenous co-expression of BARD1 and BRCA1 with NPM leads to the stabilization of NPM34. It is possible that this stabilization of NPM is due to the ubiquitylation of the protein using K6 polyubiquitin links instead of K48. NPM is a protein with contro-versial functions, as it has been assigned both oncogenic and tumour-suppressor functions35 (BOX1). However, the function of NPM in cell-cycle regulation, during development and in cancer, indicates that NPM and BRCA1–BARD1 function in a common tumour-sup-pression pathway. To some extent, NPM acts antagonis-tically to BARD1 and inhibits its BRCA1-independent functions (FIG. 3).

Another target of the BRCA1–BARD1 ubiquitin ligase is histone 2AX (H2AX). An N-terminal fragment of BRCA1, when co-expressed with BARD1, monou-biquitylates H2AX in vitro and in vivo28, which implies that BRCA1–BARD1 activity is involved in chromatin modification. This activity is reminiscent of the function of the polycomb group (PcG) of genes, which are also RING-domain-containing ubiquitin ligases36.

Transcription and DNA repair. BRCA1 can regulate gene transcription through its BRCT domains in vitro37–40. The interaction of BRCA1 with RNA polymerase (Pol) II (REF 41), RNA helicase A (REF 42) and its binding to DNA at sites of DNA branching43 also indicate that BRCA1 is involved in the regulation of transcription. BARD1 co-purifies with the BRCA1–RNA Pol II complex44, so it might function as part of this transcriptional complex, but it is not yet clear whether BARD1 has transcription activation activity.

In vitro, BRCA1–BARD1 can ubiquitylate the RNA Pol II elongation form RNA Pol IIO, mediating its deg-radation45. BRCA1–BARD1 also ubiquitylates the phos-phorylated form of the largest subunit of RNA Pol II, RNA polymerase II subunit A (POLR2A, also known as RPB1) (REF. 46). In both cases ubiquitylation is stimulated by DNA damage, and reduced by small interfering RNA (siRNA) depletion of BARD1 or BRCA1 (REF. 45). So, it is thought that the BRCA1–BARD1 ubiquitin ligase controls cell-cycle progression by targeting proteins of the stalled RNA Pol II complex.

Initial reports demonstrated that BARD1 and BRCA1 locate to nuclear dots during S phase, but relocate with RAD51 to proliferating cell nuclear antigen (PCNA)-containing structures after hydroxyurea-mediated arrest of DNA synthesis, or after UV or γ-irradiation. These findings indicate that BARD1 and BRCA1 func-tion together in DNA synthesis and repair47. BRCA1 and BRCA2 are found in complexes that are involved in homologous DNA repair and recombination48. Indeed, the BRCA1–BARD1 heterodimer has been described as the functional unit in double-strand break (DSB) repair49. BARD1 and BRCA1 also associate with other repair proteins, such as the MSH2–MSH6 complex, and this interaction indicates a role for BARD1 and BRCA1 in DNA mismatch repair50.

BARD1 also binds the mRNA polyadenylation factor cleavage stimulation factor, subunit 1, 50 kDa (CSTF1) (FIG. 3). CSTF is a protein complex required for the endonucleolytic cleavage of mRNA51,52. In vitro, BARD1 interacts with and represses the activity of the polyadenylation machinery53. The regulation of polyadenylation might be a mechanism through which BARD1 controls cellular proliferation54,55, as BARD1 binding to CSTF1 is induced by DNA damage after hydroxyurea or exposure to UV light56. However, inhi-bition of 3′ mRNA cleavage can be reversed by either BARD1 or BRCA1, or BRCA1–BARD1 depletion, indicating that degradation of RNA Pol IIO, induced by the BRCA1–BARD1 ubiquitin ligase, initiates or facilitates repair pathways by inhibiting the RNA processing machinery45.

In summary, BRCA1–BARD1 ubiquitylation path-ways influence checkpoint functions and cell-cycle arrest. In particular, centrosome duplication is control-led by γ-tubulin ubiquitylation, an anaphase checkpoint. Furthermore, BRCA1–BARD1 and BARD1 functions are physically linked to repair protein complexes. BARD1 binds and inhibits mRNA polyadenylation factor CSTF1, but this interaction depends on degradation of RNA Pol IIO through BRCA1–BARD1 ubiquitylation.

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BARD1

P P P P

p67

BARD1

BARD1

BARD1

BARD1

NF-κB

BCL3

Transcriptionregulation

Mitosis

CDK2

Transcription activators:E2FHypoxiaHormonesDNA damage

Immunogenic

Calpain cleavage

Survivalpathway

BRCA1

Deathpathway

DNAPK p53 p53

ATM

Pser15

Apoptosis

Cell-cyclearrest

DNA repairmRNA procession

RNA Pol II↓γ-Tubulin↓H2A/H2AXNPM↑

Cell-cycle controlEpigenetic control silencingApoptotic control

Ubiquitin-ligase

a

b

Oestrus, dioestrus and postoestrusOestrus specifies the time of ovulation in mammals, which can be stimulated by different factors. Dioestrus and postoestrus indicate the time before and after ovulation, respectively.

Can BARD1 function independently of BRCA1?Apoptosis. Bard1 and Brca1 are expressed in most proliferative tissues of the mouse. Interestingly, Bard1 and Brca1 expression is highest in the testes and spleen57,58 — tissues in which cells undergo high rates of proliferation and apoptosis. RNase protection assays further showed that Bard1 and Brca1 were expressed in all tissues with proliferating cells, but not the CNS58. However, in the testes, Bard1 but not Brca1 expression was found in premeiotic cells and linked to apop-tosis59. Similarly, in breast, ovarian and uterine tissue, the expression of Bard1 and Brca1 was differentially regulated in accordance with the ovulatory cycle. Specifically, Bard1 levels were increased from dioestrus to postoestrus, whereas Brca1 expression was decreased from oestrus to postoestrus58. So, BARD1 might have a role in the endometrium during the postoestrus period. This hypothesis is consistent with the finding that an inherited mutation of BARD1 is associated not only with breast and ovarian cancer but also with endometrial cancer11, an association that is not seen with BRCA1 mutations. Similarly, BARD1 is expressed

in the linings of the mammary glands, where it could be important for the control of proliferation (J.-Y. Wu and I.I.-F., unpublished observations).

BARD1 expression is also associated with apoptosis in the brain after hypoxia60. Expression of BRCA1 and BARD1 is not found in the normal brain, but expression of BARD1, in association with apoptotic markers, is seen in hypoxia induced by artery ablation60. These findings indicate that BARD1 might be expressed specifically in tissues undergoing apoptosis. Although few groups have investigated the regulation of BARD1 expression, BARD1 expression data are accumulating in various databases (for example the Gene Expression Omnibus (GEO) database).

Indeed, BARD1 is also transcriptionally upregulated in response to genotoxic stress60. Furthermore, overex-pression of exogenous BARD1 leads to apoptosis that is associated with p53 stabilization and activation of caspase 3 in various cell lines60. The apoptotic function of BARD1 is dependent on functional p53, but is inde-pendent of, and inhibited by BRCA1, as demonstrated in p53 or BRCA1-deficient cell lines60. So, BARD1 mediates

Figure 3 | Hypothetical model of BARD1 pathways and functions. BRCA1-associated ring domain 1 (BARD1) participates in two major pathways. The first is a cell survival pathway (a), mediated by the BRCA1–BARD1 heterodimer. The second (b) is a cell death pathway, which is independent of BRCA1. In pathway a, the activity of the BRCA1–BARD1 ubiqitin ligase leads to RNA Pol II degradation and cell-cycle arrest, to γ-tubulin degradation and control of centrosome duplication, to H2A/H2AX ubiquitylation and epigenetic control, and to NPM ubiquitylation and stabilization. Increased expression of NPM is known to inhibit apoptosis, and it causes centrosome amplification and genetic instability. So, NPM antagonizes BARD1 functions. In pathway b, expression of BARD1 can be increased by DNA damage, exposure to ultraviolet light, hypoxia and hormone signalling. Increased expression levels of BARD1 stabilize p53 and facilitate its phosphorylation by DNA-dependent protein kinase (DNAPK). The role of BARD1 in p53 phosphorylation at serine 15 (Pser15) by ataxia telangiectasia mutated (ATM) is unknown. Post-translational modification, through phosphorylation by CDK2–cyclin complexes, might regulate the interaction of BARD1 with BRCA1 and trigger its mitotic activity. BARD1 also has transcriptional activity as it can induce the transcription activity of NF-κBs through binding to the NF-κB co-factor BCL3. Finally, the proteolytic cleavage product of BARD1 (p67) is immunogenic and has anti-tumorigenic properties. BCL3, B cell leukaemia/lymphoma 3

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between pro-apoptotic stress pathways and the p53-dependent apoptosis pathway (FIG. 3). The importance of a BARD1-dependent apoptosis pathway is corroborated by the resistance of cells that express BARD1-antisense RNA60, and ovarian cancer cells that lack full-length BARD1 (REF. 17), to doxorubicin-induced apoptosis.

Furthermore, BARD1 has been shown to co-immu-noprecipitate with p53. The BARD1–p53 interaction leads to p53 stabilization, as the increased expression of BARD1, resulting from genotoxic stress or over-expression of exogenous BARD1, is accompanied by an increase in p53 protein levels but not mRNA levels60. The activation and stabilization of p53 is thought to depend on its phosphorylation by a number of kinases at multiple sites, including phosphorylation on serine 15 (REFS 61,62). In the absence of BARD1 (and BRCA1

(REF. 63)) phosphorylation of serine 15 is lost. BRCA1–BARD1 complexes mediate ATM and RAD3-related (ATR)-directed phosphorylation of p53, influencing G1–S cell-cycle progression after DNA damage63. BARD1 directs phosphorylation of p53-serine 15 by interacting with Ku-70, a subunit of the DNA-dependent protein kinase (DNAPK)17. Serine 15 phosphorylation of p53 is lost in cancer cells that are deficient in full-length BARD1 and resistant to induction of apoptosis, whereas overexpression of exogenous BARD1 can catalyse the phosphorylation of serine 15 (REFS 17,63) and induce apoptosis. DNAPK has an important role in the repair of double-stranded DNA breaks by a non-homologous end-joining pathway and in maintaining genomic stability. The region of BARD1 that interacts with p53 does not involve the RING-finger domain, as deletion mutants lacking this domain co-immunoprecipitate with p53 (REF. 17) (FIG. 2). The minimal region required for p53 binding spans residues 510–604, between ANK and BRCT domains, and it is this region that is required for BARD1-dependent apoptosis17,64. This region of BARD1 harbours two known cancer predisposing mutations: C557S and Q564H (REFS 11–13,64) (FIG. 1b). The Q564H mutation of BARD1 less efficiently induces apoptosis when transfected in cultured cells60, indicating that the region of BARD1 around Q564H is necessary for its tumour-suppressor and pro-apoptotic functions.

These data are consistent with the model that bind-ing of BARD1 to BRCA1 induces survival and repair functions, but an excess of BARD1 over BRCA1 leads to induction of apoptosis (FIG. 3). This hypothesis is sup-ported by experiments showing that BRCA1 reduces the apoptotic activity of BARD1, interfering with its nuclear export65. In cells deficient in BRCA1 because of inactivat-ing mutations, an excess of BARD1 over BRCA1 could induce p53-dependent apoptosis. This could explain why p53 mutations or deletions are frequent in cancers with BRCA1 mutations66. However, the status of BARD1 has not been investigated in this context.

Regulation of BARD1 expression and stability. The transcription of BARD1 is highly regulated, possibly by hormone signalling, in breast, ovarian and uterine tis-sues58, and in male germline stem cells, spermatogonia and preleptotene spermatocytes59. Two different BARD1

transcripts have been identified in these cell types — full-length BARD1 in spermatogonia and, during later stages of spermatogenesis, the splice variant BARD1β, which lacks exons 2 and 3 (that encode the RING finger domain) and exon 5 (REF. 59). This indicates that BARD1 functions are regulated during spermatogenesis by the differential expression of splice variants.

BARD1 protein stability and intracellular localization is also highly regulated26. Asynchronously growing cells show nuclear and cytoplasmic localization58,64. The human BARD1 sequence has three potential nuclear localization signal (NLS) sequences in tandem, whereas the mouse and rat sequences have three single NLS, each adjacent to one of the conserved domains of BARD1 (FIG. 1a). Deletion analyses showed that the NLS sequences in the vicinity of the ANK and BRCT domains in the mouse protein are sufficient for nuclear localization, whereas expression of the N terminus, which comprises the RING domain, only localizes to the cytoplasm64. Mutational analysis of all six NLS sequences showed that two of the non-con-served human NLS sequences are active and that the NLS sequences closest to the ANK domain are major NLS sequences in BARD1 (REF. 67) (FIG. 1). Exogenous expres-sion of mouse Bard1 deletion constructs also showed that full-length BARD1 and all constructs with the N terminus and RING domain are rapidly degraded, whereas N-ter-minal deletions that lacked the RING domain were stable. The localization into nuclear dots was dependent on the C terminus of BARD1 (REF. 64).

It was further shown that after apoptosis-induc-ing doxorubicin treatment BARD1 was degraded and appeared as a 67 kDa C-terminal fragment in the cyto-plasm. This p67 fragment retains apoptotic activity64, was previously identified in colon cancer cells, and is generated after cleavage of BARD1 by the caspase-dependent protease calpain68. Generation of the more stable p67 fragment during apoptosis might create a positive-feedback loop (FIG. 3). The injection of p67 in mice provokes an anti-tumorigenic immune response68, adding another possible tumour-suppressor function to the BARD1 repertoire (FIG. 3).

BARD1 imports BRCA1 into the nucleus, and the binding of BARD1 to BRCA1 masks the nuclear export signal (NES) in the dimerization domain of BRCA1 (REFS 26,69). The NES on BARD1 is located near the N-terminal RING domain65. Knockdown of BRCA1 expression by siRNA, or the disruption of BRCA1–BARD1 by peptide competition, caused a reduction in BARD1 nuclear localization and foci formation and an increase in the amount of BARD1 localized in the cyto-plasm, and an increase in apoptosis64,65. So, BARD1 and BRCA1 mutually control apoptosis pathways. The excess of BARD1 over BRCA1 and cytoplasmic localization is linked to induction of apoptosis by BARD1 (REF. 60), whereas nuclear retention leads to cell-cycle arrest67.

NF-κB signalling pathwaysInterestingly, BARD1 regulatory regions contain binding sites for NF-κB (C.E.J and I.I.-F., unpublished observa-tions) and might indicate that transcription of BARD1 induced by hypoxia60 is regulated by NF-κB.

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On the other hand, a role of BARD1 in the regula-tion of transcription is also plausible. The NF-κB family of transcription factors is involved in many physio-logical processes, including inflammation, apoptosis and the regulation of T- and B-cell responses, and are over expressed in many tumours70,71. BARD1 binds to B cell leukaemia/lymphoma 3 (BCL3), an NF-κB co-factor72. Specifically, the ANK repeat domain of BARD1 inter-acts with the ANK repeat domain of BCL3. BCL3 can interact with either BARD1, the histone acetylase Tip 60 (also known as HIV Tat interacting protein HTATIP), or the nuclear protein pirin, but does not bind to BRCA1 (REF. 72). Although BRCA1 is not required for the inter-action of BARD1 with BCL3, BRCA1 binds to p65/RelA, another subunit of NF-κB, and increases the transcrip-tion of NF-κB target genes, such as FAS (also known as tumour necrosis receptor superfamily, member 6 (TNFRSF6) or CD95) and interferon-β (IFNβ) (REF. 73). Whether BRCA1 depends on BARD1 for this interaction has not been determined. These data indicate that, in this instance, BRCA1 and BARD1 act in opposition to one another, as BRCA1 acts as a co-activator and BARD1 as an inhibitor of NF-κB.

Genetic instability and cell-cycle regulated expression of BARD1. The most striking feature of tumours with BRCA1 or BRCA2 deficiency is their genomic instabil-ity74. Genomic instability is also observed in embryos of Bard1 or Brca1 knockout mice16 and in cells after BARD1 repression in vitro58.

Genomic instability might be the result of deficient repair and cell-cycle control functions and an accumula-tion of damage. Chromosomal instability could arise as a consequence of defective or missing γ-tubulin ubiquit-ylation and centrosome amplification — functions that are attributed to the BRCA1–BARD1 heterodimer32. An explanation for BRCA2-dependent genomic instability is provided by the finding that depletion of BRCA2 causes cell-cycle arrest at cytokinesis owing to defective cell cleavage75. This phenotype of cells with elongated midbodies was also observed in cells with a previously described BRCA2 mutation found in breast cancer74.

Rapid acquisition of genomic instability in BARD1 depleted cells58 (C.E.J. and I.I.-F, unpublished observa-tions) argues against the hypothesis that genomic insta-bility is a consequence of accumulated damage that is due to the loss of repair functions of BARD1–BRCA1. Furthermore, the phenotype of cells depleted of BARD1 was comparable to the phenotype observed after deple-tion of BRCA2 (REF. 75), which indicates that BARD1 might function in mitosis.

BARD1, like BRCA1, is mostly expressed in prolif-erative cells, and quiescent cells show low expression levels76–80. There is evidence that BRCA1 functions dur-ing S phase81 — this is in contrast to BARD1, in which expression fluctuates in a cell-cycle-dependent manner, with maximal expression levels occurring in mitosis64,81,82. This observation is consistent with reports that BARD1 is a target for the transcription factor E2F and repressed during G0–G1, but de-repressed in RB-deficient cells83. E2F1 and E2F4 were also shown to bind to the BARD1

promotor in a high-throughput interaction screen pub-lished in the Biomolecular Interaction Network Database (BIND). So, the concomitant expression of BARD1 and BRCA1 is expected during S phase82, supporting the func-tions ascribed to the BRCA1–BARD1 heterodimer.

In addition to its cell-cycle-regulated transcription, BARD1 is phosphorylated during mitosis at its N ter-minus82, and mutagenesis of these phosphorylation sites leads to cellular hypersensitivity to mitomicin C. These data indicate that these sites might regulate BARD1 DNA repair functions in response to genotoxic stress84. BARD1 is phosphorylated by the cell-cycle dependent kinase complexes CDK2–cyclin A1/E1 and CDK2–cyclin B1 (REF. 82). One of the phosphorylation sites on BARD1 resides within the region required for the ubiq-uitin ligase activity of the BRCA1–BARD1 heterodimer (FIG. 2). Interestingly, ubiquitylation of NPM in vivo and BRCA1 auto-ubiquitylation are disrupted by co-expression of CDK2–cyclin A1/E1, but not CDK2–cyclin B1. This allows us to speculate that phosphorylation of BARD1 might interfere with BRCA1 binding and inhibit ubiquitin ligase functions.

To conclude, BARD1 is expressed in a cell-cycle-dependent manner with maximal expression in mitosis; this generates a window of overlapping expression with BRCA1 primarily during S phase. However, the increased stability of BARD1 during mitosis, and its phosphory-lation at sites that interfere with BRCA1 binding, are indicative of additional BRCA1-independent functions of BARD1 in mitosis. The role of BARD1 in mitosis seems to be a dual one; a BRCA1–BARD1 function that controls centromere replication through the ubiquity-lation of γ-tubulin and a BRCA1-independent function is suspected, as BARD1 stabilization during mitosis is induced by phosporylation at sites that interfere with BRCA1 binding82 (FIGS 2,3).

Polycomb genesThe numerous functions of BARD1 and BRCA1– BARD1 are reminiscent of the functions of the PcG gene family. Homozygous mutations in PcG genes cause embryonic lethality, and heterozygous mutations con-fer homeotic transformation. So, these mutations have been classified as dominant. PcG proteins are repres-sors of genes of the bithorax complex. In mammalian systems, at least two groups of PcG proteins are defined. These are implicated in initiation and maintenance of silenced states of chromatin (TABLE 1). Similar to PcG proteins, BARD1 and BRCA1 have RING fingers and long stretches of protein sequence with little secondary structure and no homology to any other known pro-tein. Heterozygous mutations in PcG genes predispose human cells to transformation. In addition, there is evi-dence that both BRCA1 and BRCA1–BARD1 interact with chromatin-modification complexes, much like the PcG proteins do (TABLE 1).

This comparison allows us to speculate as to why BRCA1 and BRCA2 were discovered as cancer pre-disposition genes, but BARD1 was not. BRCA1 muta-tions in human cells (much like PcG mutations in Drosophila) do not immediately result in a malignant

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(transformed) phenotype, but have a cumulative effect that is possibly caused by incorrect stoichiometry with other interacting proteins and complexes. Mutations in BARD1 might be more deleterious than BRCA1 muta-tions because of the additional apoptotic and potential mitotic functions of BARD1, but carcinogenesis might be facilitated in cells that survive BARD1 disruption. Indeed, one explanation of why BRCA1 mutations are associated with cancers in hormone-dependent tis-sues is based on the hypothesis that BRCA1 deficiency is tolerated without being lethal for the cells, owing to the anti-apoptotic action of oestrogens in these tissues85.

Future directionsBARD1 has multiple functions, but its cancer-asso-ciated activities seem to centre on two major path-ways: as an essential component of a ubiquitin ligase involved in DNA repair and cell-cycle regulation, and in p53-mediated apoptosis. Its BRCA1-dependent function resides at its N terminus, whereas its apop-tosis-mediating function lies within its C terminus. The C terminus also harbours the germline mutations found in cancer patients. In contrast to findings for

BRCA1, no BARD1 mutations are reported within the BRCA1-interaction domain. These data indicate that BARD1 possesses other functions in addition to its role as a BRCA1-related tumour suppressor. Furthermore, tumours seem to express truncated forms of BARD1. These forms lack domains that regulate p53 function and apoptosis, or other as yet unidentified functions. The expression of these truncated forms of BARD1 correlates with a poor prognosis and so these forms might prove to be useful prognostic indicators and/or tools for breast cancer screening.

BARD1 is also required for proliferation, and loss of BARD1 expression rapidly causes genomic instability leading to cell death. Cells that survive in the absence of BARD1 are therefore primed for malignant transformation. It will be important to find out whether BARD1 repression might occur by epigenetic changes during tumorigenesis. This can be determined by examining BARD1 expression levels in premalignant tissue. Answering questions such as these, and identifying the factors and/or mechanisms that govern the repression of functional BARD1, will identify many new components of breast and ovarian cancer pathogenesis.

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3. Wu, L. C. et al. Identification of a RING protein that can interact in vivo with the BRCA1 gene product. Nature Genet 14, 430–440 (1996).The BRCA1 N-terminal region was used as bait in a two-hybrid interaction screen. BARD1 emerged as the major interacting protein, and mutations in

BRCA1 inhibited this interaction. BARD1 shares similarities with BRCA1 at the N terminus, possessing a RING-finger domain, and at the C terminus. The C-terminal homologous domains, which contain unusual protein motifs, are structurally similar to protein regions in many

Table 1 | Similarities between PcG, BRCA1–BARD1 and BRCA2 function

Function PRC1 PRC2 BRCA1–BARD1 BRCA2

Maintenance of gene expression state

Yes, maintains gene expression

Yes, initiates gene silencing

Yes Yes, interacts with CBP, which is involved in regulating gene expression

Function as histone methyltransferases

No Yes No No

Fuction as histone acetylase No No No Yes (REF. 91)

Function in X inactivation Yes Yes (REF. 87) Yes, interacts with XIST (REF. 90)

No

Chromatin remodelling by the SWI–SNF complex

Yes, negatively regulates this process (REF. 88)

No No Yes, interacts with SNF

Transformation Mammalian BMI1 involved in cellular transformation

Yes Yes Yes

Stem-cell regulation BMI1 regulates haematopoietic stem cells (REF. 89)

Not known BARD1 implicated in the regulation of germline stem cells (REF. 59)

No

Cell-cycle control Yes Yes Yes Yes

DNA repair function No No Yes YesPcG proteins are known to be part of a memory system that ensures the faithful transmission of cell identities through cell division92. PcG protein complexes in Drosophila melanogaster (PRC1 and PRC2) comprise Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs), Esc (Extra sex combs), E(z) (Enhancer of zeste), and Su(z) (Suppressor of zeste), for which homologues were found in mammals as were other components of the complexes. This table highlights the similarities between the function of the polycomb group of genes in flies and mammals with the known functions of BRCA1, BRCA2 and BRCA1-associated ring domain 1 (BARD1). The data indicate that they could be functionally similar. CBP, CREB binding protein; SWI–SNF, chromatin remodelling complex conserved between yeast, flies and mammals; XIST, X-inactivation specific transcript.

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AcknowledgementsI am grateful to G.J. Laurent and W.C. Leung for discussions, comments and critiques. This work was supported by a grant from the Swiss National Science Foundation (SNSF) to I.I.-F.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBRCA1 | BRCA2 | IFNβ | TNFRSF6Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=proteinBARD1 | BCL3

FURTHER INFORMATIONBiomolecular Interaction Network Database BIND: http://bind.ca/Action?pg=3001&identifier=bindid&idsearch=194856Genecard: http://www.genecards.orgGene Expression Omnibus (GEO) database: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geoHuman Gene Mutations Database BIOBASE: http://www.hgmd.cf.ac.ukSearch Tool for the Retrieval of Interacting Genes/Proteins STRING: http://string.embl.de/newstring_cgi/show_input_page.pl?Simple Modular Architecture Research Tool (SMART): http://smart.embl-heidelberg.deAccess to this links box is available online.

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Is there more to BARD1 than BRCA1?

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