Yanfen Hu

BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations

The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator–containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5′ mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3′ region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions.

Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression

Mutations in BRCA1 increase risks of familial breast and ovarian cancers, particularly among premenopausal women. While BRCA1 plays an active role in DNA repair, this function alone may not be sufficient to explain why BRCA1-associated tumors predominantly occur in estrogen-responsive tissues. Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Aromatase expression in ovarian granulosa cells dictates levels of circulating estrogen in premenopausal women, and its aberrant overexpression in breast adipose tissues promotes breast cancer growth. Here, we show that BRCA1 modulates aromatase expression in ovarian granulosa cells and primary preadipocytes. The cyclic AMP-dependent expression of aromatase in ovarian granulosa cells is inversely correlated with the protein level of BRCA1. Importantly, transient knockdown of BRCA1 enhances aromatase expression in both ovarian granulosa cells and primary preadipocytes. We propose that BRCA1 deficiency in epithelial and certain nonepithelial cells may result in combined effects of aberrant estrogen biosynthesis and compromised DNA repair capability, which in turn may lead to specific cancers in the breast and ovary.

An activation-independent role of transcription factors in insulator function

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti‐silencing regions behave as boundaries to telomere‐driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Characterization of a Novel Trans-Activation Domain of BRCA1 That Functions in Concert with the BRCA1 C-terminal (BRCT) Domain

Mutations in the breast cancer susceptibility gene, BRCA1, account for a significant proportion of hereditary breast and ovarian cancers. The BRCA1 C-terminal (BRCT) domain, which can activate transcription when fused to a heterologous DNA binding domain, is required for BRCA1 function in suppression of tumorigenesis. Here, we provide evidence for a new activation domain in BRCA1 that lies adjacent to the BRCT domain. We name the two domains AD1 and AD2, respectively. Like AD2, the newly discovered AD1 can act independently as an activation domain in both yeast and human cells. However, unlike AD2, AD1 activity in mammalian cells is cell type context-dependent. Furthermore, combination of these two domains in mammalian cells can result in a robust synergy in transcriptional activation. A highly conserved coiled-coil motif in AD1 is required for the cooperative transcription activation. Interestingly, the functional cooperativity between AD1 and AD2 is absent in certain breast and ovarian cancer cell lines, although each domain can still activate transcription. Therefore, the differential and cooperative actions of the two activation modules may contribute to the heterogeneous risk of BRCA1 mutations in different tissues.

A phosphotyrosine switch determines the antitumor activity of ERβ

Estrogen receptors ERα and ERβ share considerable sequence homology yet exert opposite effects on breast cancer cell proliferation. While the proliferative role of ERα in breast tumors is well characterized, it is not clear whether the antitumor activity of ERβ can be mobilized in breast cancer cells. Here, we have shown that phosphorylation of a tyrosine residue (Y36) present in ERβ, but not in ERα, dictates ERβ-specific activation of transcription and is required for ERβ-dependent inhibition of cancer cell growth in culture and in murine xenografts. Additionally, the c-ABL tyrosine kinase and EYA2 phosphatase directly and diametrically controlled the phosphorylation status of Y36 and subsequent ERβ function. A nonphosphorylatable, transcriptionally active ERβ mutant retained antitumor activity but circumvented control by upstream regulators. Phosphorylation of Y36 was required for ERβ-mediated coactivator recruitment to ERβ target promoters. In human breast cancer samples, elevated phosphorylation of Y36 in ERβ correlated with high levels of c-ABL but low EYA2 levels. Furthermore, compared with total ERβ, the presence of phosphorylated Y36-specific ERβ was strongly associated with both disease-free and overall survival in patients with stage II and III disease. Together, these data identify a signaling circuitry that regulates ERβ-specific antitumor activity and has potential as both a prognostic tool and a molecular target for cancer therapy.

Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element

Estrogen is critical to both normal mammary gland and breast cancer development. Circulating levels of estrogen in premenopausal women are primarily determined by the action of aromatase in ovarian granulosa cells that converts testosterone to estradiol. In the current study, we unraveled an important role of Jun proteins in modulating ovary-specific aromatase expression. Ectopic expression of the Jun proteins in a human granulosa cell line significantly inhibited an ovary-specific promoter (PII) of the aromatase gene, whereas expression of dominant-negative mutants of Jun led to increased promoter activity. The Jun-mediated repression was specific to the aromatase promoter, as Jun proteins stimulated known AP1-responsive promoters in the same cellular context. Both the activation and basic leucine zipper domains of Jun were required for the transcriptional repression. Electrophoretic gel mobility assay showed that endogenous Jun proteins bound to a functionally important cAMP-responsive element (CRE) in the PII promoter-proximal region. Alteration of the CRE-like site impaired both the cAMP-responsive transcriptional activation and Jun-mediated repression. Furthermore, chromatin immunoprecipitation indicated the presence of cJun at the proximal region of the native PII promoter. Taken together, our work suggests that Jun proteins may attenuate estrogen biosynthesis by directly downregulating transcription of the aromatase gene in ovarian granulosa cells.

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

Background: Lower level of breast cancer suppressor BRCA1 has been reported in sporadic breast cancers. Results: SCFFBXO44 E3 ligase controls BRCA1 stability and is overexpressed in many sporadic breast cancers with low BRCA1 level. Conclusion: SCFFBXO44-mediated BRCA1 stability may contribute to sporadic breast cancer development. Significance: Regulation of BRCA1 stability provides new insights and targets on BRCA1-mediated sporadic breast cancer. BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCFFBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCFFBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCFFBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCFFBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

Regulation of adipose oestrogen output by mechanical stress

Adipose stromal cells are the primary source of local oestrogens in adipose tissue, aberrant production of which promotes oestrogen receptor-positive breast cancer. Here we show that extracellular matrix compliance and cell contractility are two opposing determinants for oestrogen output of adipose stromal cells. Using synthetic extracellular matrix and elastomeric micropost arrays with tunable rigidity, we find that increasing matrix compliance induces transcription of aromatase, a rate-limiting enzyme in oestrogen biosynthesis. This mechanical cue is transduced sequentially by discoidin domain receptor 1, c-Jun N-terminal kinase 1, and phosphorylated JunB, which binds to and activates two breast cancer-associated aromatase promoters. In contrast, elevated cell contractility due to actin stress fibre formation dampens aromatase transcription. Mechanically stimulated stromal oestrogen production enhances oestrogen-dependent transcription in oestrogen receptor-positive tumour cells and promotes their growth. This novel mechanotransduction pathway underlies communications between extracellular matrix, stromal hormone output, and cancer cell growth within the same microenvironment.

IKKβ Mediates Cell Shape-Induced Aromatase Expression and Estrogen Biosynthesis in Adipose Stromal Cells

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in breast cancer therapy. Within tumor microenvironment, tumor cells stimulate aromatase expression in adipose stromal cells (ASCs), which in turn promotes estrogen-dependent growth of estrogen receptor (ER)-positive tumor cells. However, it is not clear how aromatase transcription and estrogen biosynthesis are regulated in ASCs under a precancerous condition. Here we demonstrate that cell shape change alone is sufficient to induce aromatase expression in primary ASCs from cancer-free individuals. The activation of aromatase transcription is mediated by IkappaB kinase-beta (IKKbeta), a kinase previously known for its cancer-promoting activity in tumor cells. Activation of IKKbeta leads to elevated expression of transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta), which binds to and stimulates two breast cancer-associated promoters of the aromatase gene. We also show that shape-induced estrogen production in ASCs can stimulate estrogen-dependent transcription in ER-positive breast tumor cells. We suggest that IKKbeta-dependent aromatase induction due to changes in cellular architecture in adipose tissue may contribute to the breast cancer risks associated with high mammagraphic density and obesity.

BRCA1 transcriptional activity is enhanced by interactions between its AD1 domain and AhR.

PurposeWe previously reported that BRCA1 interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) and that this interaction affects TCDD-induced CYP1A1 gene expression (Kang et al., J Biol Chem 281:14654–14662, 2006). In this study we continue this investigation and begin to define the significance of this interaction for the regulation of stress-induced transcription.MethodsImmunoprecipitations (IPs), western blot (WB) analysis, GST pull-down assays and promoter reporter assays were used to investigate whether the aryl hydrocarbon receptor (AhR) can regulate transcription that is dependent on the activation domain 1 (AD1) domain of BRCA1.ResultsWe show that AhR, a transcription factor, can bind specifically to AD1 in the C-terminal region of BRCA1 and affect BRCA1’s ability to regulate transcription activity. We found that xenobiotics that positively and negatively affect AhR’s activity as a transcription factor (e.g., dioxin and α-naphthoflavone, respectively), have similar effects on AhR’s ability to affect AD1-domain-dependent transcription. These physical and functional AhR–AD1 interactions may require the coiled-coil motif in AD1 because point-mutations in this motif reduce these interactions.ConclusionXenobiotic-activated AhR can function in two ways, as a component of the AhR/ARNT transcription factor and a regulator of AD1-dependent transcription. Consequently, BRCA1 has two distinct mechanisms for sensing xenobiotics and regulating AhR-dependent stress responses to these xenobiotics. We speculate that the normal functioning of this interaction could play a role in BRCA1’s tumor suppressing ability.