ACCELERATED PUBLICATION
BRCA1 Is a Selective Co-activator of 14-3-3sigma Gene Transcription in Mouse Embryonic Stem Cells*,

Olga AprelikovaDagger , Amy J. Pace§, Bruno FangDagger , Beverly H. Koller§, and Edison T. LiuDagger

From the Dagger  Section of Molecular Signaling and Oncogenesis, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland, 20892 and the § Curriculum in Genetics and Molecular Biology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received for publication, May 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

BRCA1 gene is a tumor suppressor for breast and ovarian cancers with the putative role in DNA repair and transcription. To characterize the role of BRCA1 in transcriptional regulation, we analyzed gene expression profiles of mouse embryonic stem cells deficient in BRCA1 using microarray technology. We found that loss of BRCA1 correlated with decreased expression of several groups of genes including stress response genes, cytoskeleton genes, and genes involved in protein synthesis and degradation. Previous study showed that BRCA1 is a transcriptional co-activator of p53 protein; however the majority of p53 target genes remained at the same expression levels in BRCA1 knockout cells as in the wild type cells. The only p53 target gene down-regulated with the loss of BRCA1 was 14-3-3sigma , a major G2/M checkpoint control gene. Similar to cells with decreased 14-3-3sigma activity, BRCA1-deficient cells were unable to sustain G2/M growth arrest after exposure to ionizing radiation. We find that BRCA1 induction of 14-3-3sigma requires the presence of wild type p53 and can be regulated by a minimal p53 response element.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutations in BRCA1 gene are linked to inherited breast and ovarian cancers. The biological function of BRCA1 is not clearly understood. Primary cells deficient in BRCA1 have decreased growth rate, altered chromosome stability, and G2/M checkpoint control (1-5).

Evidence suggests that BRCA1 is involved in a number of DNA repair pathways, with BRCA-/- embryonic stem (ES)1 cells being impaired in their ability to perform homologous recombination and transcription-coupled repair after oxidative DNA damage (6-8). After DNA damage BRCA1 protein is phosphorylated and activated by ATM, ATR, and Chk2 protein kinases, major regulators of DNA damage response (9-11). This phosphorylation initiates relocation of the BRCA1 protein inside the nuclei to the sites of DNA repair and most likely ensures its interaction with other proteins implicated in homologous recombination and double-strand break repair (12, 13).

Another putative function of BRCA1 is in the regulation of transcription. BRCA1 interacts with a number of transcription factors or modifiers of transcription like p53, c-Myc, CtIP·CtBP, STAT1, and p300 (14-19). Binding to different transcription factors results in a diverse biological outcome. For example, BRCA1 induces the transcription of cell cycle regulators cyclin-dependent kinase inhibitor p21 and GADD45 by virtue of p53 transcriptional co-activation or represses the expression of the same genes when silencing ZBRK1 transcription factor through the CtIP·CtBP complex (20). Mutations identified in the C terminus of BRCA1 from breast cancer patients were unable to activate transcription, inferring the importance of transcriptional activating function for BRCA1 tumor suppression activity.

To characterize the role of BRCA1 in transcriptional regulation, we analyzed gene expression profiles of ES cells with targeted deletion of full-length BRCA1 gene. We found that loss of BRCA1 results in decreased expression of stress response genes, cytoskeletal genes, and 14-3-3sigma , a major G2/M checkpoint control gene. Irradiation of BRCA1-deficient cells showed the preliminary exit from G2/M growth arrest similar to cells with targeted deletion of 14-3-3sigma (21). BRCA1 protein overexpression activated the 14-3-3sigma promoter or induced transcription of the endogenous 14-3-3sigma in a p53-dependent manner.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Reporter Plasmids-- The 14-3-3sigma promoter cloned upstream of luciferase gene was a gift of Dr. B. Vogelstein (Johns Hopkins University) and is described in Ref. 28, and p53 wild type, mutant p53-273H, and pG13-luc were obtained from Dr. Kevin Gardner (NCI, NIH, Gaithersburg, MD). PcDNA3-BRCA1 wild type was a gift of Dr. M. Erdos (NHGRI, NIH, Bethesda, MD). PcDNA3-BRCA1Delta C has a deletion of 94 C-terminal amino acids. PcDNA3.1/His/lacZ plasmid was purchased from Invitrogen (Carlsbad, CA).

Cell Cultures-- Generation of ES cells with the targeted deletion of exon 11 of the BRCA1 gene and BRCA1-deficient ES cells transfected with a BRCA1 transgene were described in Refs. 1 and 8. Mcf7 breast carcinoma, HCT116, RKO, SW480, and HT29 colon cancer cell lines were purchased from American Type Culture Collection and cultured in recommended growth media. HCC1937 cells were a gift from Dr. Mel Campbell and Dr. Roy Jensen (Vanderbilt University, Nashville, TN). Cells were irradiated using a 137Cs gamma -irradiator at total dosage of 10 Gy.

Microarray Analysis-- Messenger RNA was isolated from ES cells using a FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). 2 µg of mRNA was reverse transcribed using T7(dT)24 primer and then labeled with biotinylated ribonucleotides incorporated by T7 RNA polymerase. Resultant RNA was fragmented and hybridized to an oligonucleotide microarray Mu6500 chip (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations. BRCA1-/- mRNA hybridization was repeated twice, and data from each hybridization were used as a baseline for comparison with BRCA1+/+ and BRCA1-tg cells. Comparison of gene expression was performed with GeneChip analysis software. Signal intensities for different chips were scaled to the target value of 150 for GAPDH gene (3' and M probes). The genes were excluded from the list of outliers if the decision algorithm used negative values of average differences. Only genes reproduced in 3 of 4 cross-comparisons resulting in more than 2-fold difference were considered as true outliers.

Northern Blot Analysis-- Total RNA was isolated using Trizol Reagent (Life Technologies, Inc.), and 20 µg was used for analysis. A probe containing the 3'-untranslated region of the mouse 14-3-3sigma gene was generated by polymerase chain reaction using expressed sequence tag AA873962 (Research Genetics, Huntsville, AL) and M13 forward and reverse primers. A 14-3-3sigma human probe was also generated by polymerase chain reaction from the 3'-untranslated region as described in Ref. 26. To generate mouse BRCA1 probe, an internal 4.5 kilobase pair EcoRI fragment was isolated. Hybridizations were performed in QuickHyb solution using the manufacturer's instructions (Stratagene).

Flow Cytometry-- ES cells were gamma -irradiated with a dose of 10 Gy and collected at the indicated time points. 106 cells were fixed with 70% ethanol and stained with propidium iodide, and amounts of DNA per cell were determined using FACScan (Becton Dickinson).

Infection with Adenoviruses-- The amount of virus for infection was variable (50 multiplicity of infection/cell for HCT116 or 500 multiplicity of infection/cell for Mcf7 and RKO) to provide about two times overexpression of BRCA1. 16 h after infection total RNA was analyzed for 14-3-3sigma or BRCA1 expression by Northern blotting.

Luciferase Reporter Assay-- Cells were split 24 h prior to the experiment into 60-mm plates and transfected at about 60% density with LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's instructions. Briefly, each transfection reaction contained 0.5 µg of luciferase reporter and 0.2 µg of lacZ plasmid as an internal standard. Where indicated cells were transfected with 1 µg of p53 expression vector (or mutant p53-273H) and 12 µg of pcDNA3-BRCA1 (or pcDNA3-BRCA1Delta C). The total amount of DNA was balanced with equimolar amount of empty vector. In 24 h cells were washed with phosphate-buffered saline, scraped into 400 ml of 0.25 M Tris, pH 8.0, and snap frozen in dry ice. After two rounds of freeze-thaw cellular lysates were cleared by centrifugation, 10 µl of lysate was tested for beta -gal activity using a beta -gal assay kit (Invitrogen, Carlsbad, CA), and 5-10 µl of lysate was used to measure luciferase activity (luciferase assay system, Promega, Madison, WI).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Differential Gene Expression in BRCA1-deficient Cells-- To identify the impact of loss of BRCA1 function, we compared the gene expression in the following three cell lines: wild type ES cells, ES cells deficient in BRCA1 expression as a result of targeted mutation of exon 11 (these cells will be termed BRCA1-/-), and BRCA1-/- ES cells reconstituted by transfection with a mouse BRCA1 cDNA expression vector, transgene. Amplified mRNA from these cells was hybridized to an oligonucleotide microarray (Mu6500; Affymetrix). We considered as outliers the genes whose altered expression in BRCA1-/- cells compared with BRCA1+/+ cells was at least partially restored after ectopic expression of a BRCA1 transgene in the BRCA1-/- cells. Some BRCA1 regulated genes may not have achieved outlier status in our list, because the level of BRCA1 transgene expression was lower than that in the wild type cells, and therefore the reconstitution of BRCA1 expression was not complete (see Fig. 1A and Ref. 7). Nevertheless, this approach allowed us to increase the reliability of the results increasing the probability that the expression of these genes is directly or indirectly regulated by BRCA1.


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Fig. 1.   14-3-3sigma is down-regulated in BRCA1-/- cells. A, Northern blot analysis of 14-3-3sigma and BRCA1 gene expression in the parental embryonic stem cells (+/+), cells deficient in BRCA1 (-/-), and -/- cells transfected with BRCA1 cDNA expression vector (tg). B, induction of the 14-3-3sigma gene in ES cells after irradiation. BRCA1+/+ and -/- cells were irradiated at 10 Gy, and the amount of 14-3-3sigma transcript was analyzed by Northern blotting. Average data from three independent experiments is presented. BRCA1+/+, open circle ; BRCA1-/-, . C, cell cycle analysis of BRCA1+/+, BRCA1-/-, or BRCA1-tg cells after exposure to 10-Gy irradiation. Cells were collected at different time points after irradiation and analyzed by flow cytometry. The amount of cells in each phase of the cell cycle is presented in the right panel. BRCA1+/+, solid line; BRCA1-/-, thin line; BRCA1-tg, dotted line.

Of the 6,500 gene elements represented in the array, 51 genes were expressed at more than two times higher levels in the presence of either the wild type or the reconstituted BRCA1, whereas no genes were down-regulated by BRCA1 within the stringent definition of outliers (see Table I in Supplemental Material). Expression of several genes involved in regulation of transcription was reduced with BRCA1 deletion. Included in this group is Id3, a dominant negative inhibitor of transcription. Id3 gene is induced during early G1 phase, following by a second peak of induction in the late G1 or early S phase coincident with the expression of BRCA1. Partial inactivation of Id3 protein by antisense oligonucleotide or antibody microinjection results in a delayed entry of cells into the S phase of cell cycle. Similar to BRCA1, overexpression of Id3 induces apoptosis (22). Using Northern blot analysis we have confirmed that both the wild type and BRCA1-tg ES cells express higher levels of Id3 gene than BRCA1-/- cells (data not shown).

Loss of the BRCA1 resulted in reduced expression of a diverse group of genes involved in cytoskeleton reorganization including multiple forms of alpha -actin, ROCK kinase, stabilizing stress fibers, cytokeratin, vimentin, and tropomyosin. Cell cycle progression is associated with dramatic changes in the organization of cytoskeletal filaments. Expression of these genes is induced during S-G2/M phase of cell cycle (23) and repressed during oncogenic transformation. Similar to the study reported here, microarray analysis of genes induced by p53 revealed up-regulation of genes encoding cytoskeletal proteins (24). We also found decreased expression of the Pw1 gene in BRCA1-/- cells. This gene is activated during p53/c-Myc-mediated apoptosis but not during p53-dependent G1 arrest, suggesting that Pw1 cooperates with p53 in determining the choice between cell death and survival (25). A number of the genes with altered expression are involved in protein synthesis and degradation, as well as heat shock genes, whose products are known to modify protein folding in response to cellular stress.

Down-regulation of 14-3-3sigma Gene in BRCA1-/- Cells-- We consistently found decreased expression of 14-3-3sigma in the BRCA1-deficient cells. Northern blot hybridization of RNA prepared from the three cell lines confirmed the differences obtained on analysis of our microarray data (Fig. 1A). In addition, this induction is specific for 14-3-3sigma in that other 14-3-3 family members did not change with the BRCA1 status. Previous studies have shown that 14-3-3sigma gene is induced after genotoxic stress (26). We, therefore, determined whether expression of BRCA1 could modify the cellular response to gamma -irradiation using 14-3-3sigma expression as the read out. Our results show that induction of 14-3-3sigma transcripts is significantly depressed in BRCA1-/- ES cells after exposure to 10 Gy of gamma -irradiation (Fig. 1B).

BRCA1-/- Cells Exit G2/M Growth Arrest Prematurely-- The 14-3-3sigma gene product is an important mediator of G2/M checkpoint control, blocking progression of cells with DNA damage through mitosis by retention of the CDC2·cyclin B complex in the cytoplasm (21). Therefore, 14-3-3sigma -/- cells arrest in G2/M phase of the cell cycle after exposure to DNA-damaging agents but exit this arrest prematurely. We determined whether the loss of BRCA1 and consequent decreased expression of 14-3-3sigma leads to similar alteration of cell cycle regulation in ES cells and whether these changes could be restored by expression of the BRCA1 transgene. Wild type, BRCA1-/-, and BRCA1-tg cells were irradiated, and cell cycle progression was measured over the next 24 h. ES cells do not display an arrest in G1. Thus, after exposure to irradiation, the G1 population decreased whereas the number of cells in G2 increased dramatically. After 4 h, all three ES cell lines had a similar arrest in G2/M (Fig. 1C). However, even at 8 h post-irradiation, the number of BRCA1-/- cells in G2/M phase of cell cycle was significantly lower than that in parental BRCA1+/+ or BRCA1-tg cells. Moreover, the premature reentry of the BRCA1-/- cells into the cell cycle was observed by the increase in the number of cells in G1 at the later time points. This pattern of behavior strikingly resembles that of 14-3-3sigma -/- cells and suggests a functional consequence of the attenuated 14-3-3sigma expression in the BRCA1-disrupted ES cells after DNA damage.

In the previous study, p53 was identified as a transcriptional factor responsible for 14-3-3sigma induction after DNA damage (26). Therefore, we examined the status of p53 expression in BRCA1-/- cells. Neither microarray nor Northern blot analysis revealed any difference in levels of p53 transcript between these cell lines. Analysis of p53 protein by Western blot showed no differences in protein level or in phosphorylation of serine in position 15 of p53 in untreated wild type or BRCA1-/- cells (data not shown). 8 h after irradiation, phosphorylation of p53, as well as p21 protein, rose to similar levels in the parental and BRCA1-deficient lines. These results show that the decreased expression of 14-3-3sigma in BRCA1-/- cells is not associated with changes in the p53 protein.

Induction of 14-3-3sigma by BRCA1 Overexpression in p53-positive Human Cells-- To obviate the possibility that decreased expression of 14-3-3sigma gene may be unique to mouse ES cells used in our microarray experiments, we infected several human cancer cell lines with adenovirus encoding the BRCA1 gene. The 14-3-3sigma transcript was induced in all three cancer cell lines, harboring wild type p53 genes (Fig. 2A). Isogenic virus encoding truncated BRCA1 protein was significantly less potent in inducing 14-3-3sigma expression (Fig. 2B). When we used the colon cancer cell line SW480, which is deficient in p53 protein, or the breast carcinoma cell line HCC1937, which is deficient in both p53 and BRCA1, no increase in 14-3-3sigma transcription was observed (Fig. 2C). Somasundaram et al. (27) reported that BRCA1 overexpression results in an increase in p53 protein levels 24 h after infection with adenovirus, and this increase is dependent on p14ARF. To verify that 14-3-3sigma induction after adenovirus-mediated BRCA1 overexpression was not a secondary reaction because of p53 protein stabilization we monitored the amount of 14-3-3sigma transcript and levels of p53 and BRCA1 proteins at various time points after infection. (Fig. 3A). The BRCA1 protein was easily detectable 8 h after infection, and induction of 14-3-3sigma transcripts expression was observed within the first 12 h of the experiment. In contrast, p53 induction could not be measured until at least 20 h post-infection. Together with previous data this observation suggests that whereas p53 is required for induction of 14-3-3sigma , BRCA1 can modulate expression of this gene.


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Fig. 2.   Northern blot analysis of 14-3-3sigma induction by BRCA1 overexpression in human cells. A, p53-positive mammary carcinoma cell line Mcf7 or colon cancer cell lines HCT116 and RKO induced 14-3-3sigma gene expression after infection with adenovirus encoding BRCA1 compared with empty virus (AE1). B, HCT116 cell line was infected with adenovirus encoding wild type BRCA1 gene or mutated BRCA1 (1853-Stop) deficient in transcriptional activation. C, p53-negative colon cancer cell line SW480 and BRCA1 and p53 negative breast carcinoma cell line HCC1937 did not induce 14-3-3sigma after adenovirus-mediated BRCA1 overexpression. Moi, multiplicity of infection.


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Fig. 3.   BRCA1 cooperates with p53 in stimulation of 14-3-3sigma expression. A, Northern blot analysis of 14-3-3sigma at different time after adenovirus-mediated BRCA1 overexpression in HCT116 cell line. GFP, green fluorescent protein. B, Western blot analysis of p53 and BRCA1 protein at different time points after BRCA1 overexpression in HCT116 cell line. C, BRCA1 stimulates p53-dependent transcription from the 14-3-3sigma promoter. HT29 colon cancer cell line was co-transfected with the 14-3-3sigma reporter plasmid and pcDNA3-lacZ, as an internal standard, and combinations of p53, mutant p53-273H and BRCA1, or transactivation-deficient BRCA1 mutant. Cells were harvested 24 later, and luciferase and beta -gal activity were measured. D, as a control, p53 artificial promoter pG13 (15) was used instead of the 14-3-3sigma promoter. Experiments were repeated at least three times in duplicate. Representative experiments are shown.

The ability of BRCA1 to modify 14-3-3sigma expression was further tested by examining transcription of a luciferase gene driven by the 14-3-3sigma promoter. This reporter plasmid was co-transfected with BRCA1 and/or p53 expression vectors into two p53-negative colon cancer cell lines, HT29 and SW480 (Fig. 3B and data not shown). As expected, p53 gene alone induces luciferase gene expression. In contrast, BRCA1 alone had a very minor effect on the activity of the 14-3-3sigma promoter. However, when both p53 and BRCA1 were co-expressed, transcription was induced to significantly higher levels than that observed in cells transfected with p53 alone. The magnitude of induction was comparable with that of an artificial promoter comprised of 13 tandem p53 response elements (Fig. 3C). To further verify the role of BRCA1 in increased activity of the 14-3-3sigma promoter, we compared the effectiveness of wild type and a mutant BRCA1 in this assay. We found that a C-terminal truncation mutant of the BRCA1 was defective in its ability to co-activate transcription from the 14-3-3sigma promoter. Our findings indicate that BRCA1 alone is a very poor transcriptional activator of the 14-3-3sigma but that BRCA1 can act synergistically to augment the ability of p53 to induce the transcription of the 14-3-3sigma gene.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The focus of this work is to characterize the changes in the gene expression in cells with targeted deletion of BRCA1 exon 11. Of the list of 51 genes induced by BRCA1, we focused on 14-3-3sigma gene because of its role in cell growth control. The importance of 14-3-3sigma protein in mammary cell transformation is suggested by the fact that 14-3-3sigma expression is silenced in the majority of breast cancers (28). Previous reports have suggested that BRCA1 is a transcriptional co-activator of p53. However, we find that BRCA1 is not a broad co-activator but rather a selective modulator of p53 transcription activity. The majority of p53 target genes presented in the chip did not alter their transcript levels with the loss of BRCA1. Obviously the most interesting question to address in the future will be to learn what other factors influence the BRCA1 choice of transcriptional activation.

Previous microarray analysis of gene expression in the U2OS osteosarcoma cell line after induction of the BRCA1 protein did not show 14-3-3sigma gene to be BRCA1 responsive (29). This may be explained by cellular specificity of the 14-3-3sigma , which is expressed mainly in epithelial cells. In agreement with this, we did not see induction of 14-3-3sigma in U2OS cells treated with adriamycin, unlike several breast or colon cancer cell lines (data not shown). Another study of downstream BRCA1 target genes focused mainly on p53-negative cells, and the inability of BRCA1 to induce 14-3-3sigma gene expression confirms that BRCA1 acts through p53 (30). Of note, both publications were utilizing the overexpression of BRCA1 above the endogenous levels, which may explain the differences in the set of genes activated by BRCA1.

Our results show that the expression of the 14-3-3sigma checkpoint control gene is modulated by the BRCA1 status of a cell. Moreover, optimal expression of 14-3-3sigma after DNA damage can be induced by a synergistic effect between BRCA1 and p53. These data imply that depending on the cellular context, BRCA1 can be a limiting factor in maximal p53-dependent transactivation. Our findings might also explain several clinical observations. First, there is increasing information linking the loss of 14-3-3sigma expression to epithelial transformation. Given the synergistic interaction between BRCA1 and p53 in regulating 14-3-3sigma expression described here, haploinsufficiency in either BRCA1 or p53 genes might contribute to an inadequate 14-3-3sigma response to DNA damage and ultimately to transformation of breast epithelium. Second, our observation suggests that mutation in BRCA1 gene would compromise the 14-3-3sigma function, which is operative mainly in epithelial cells. Thus, despite the ubiquitous expression of BRCA1, its regulation of 14-3-3sigma expression might explain the epithelial specificity of cancers in BRCA1 mutation carriers.

    ACKNOWLEDGEMENTS

We thank Dr. Vert Vogelstein for 14-3-3sigma reporter, Dr. Kevin Gardner for p53 and p53-273H expression vectors, and Dr. Mel Campbell and Dr. Roy Jensen for adenoviruses.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA82423 (to B. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table I.

To whom correspondence should be addressed: ATC Bldg., Rm. 121, 8717 Grovemont Cr., Gaithersburg, MD 20878. Tel.: 301-435-5774; Fax: 301-402-3134; E-mail: apreliko@mail.nih.gov.

Published, JBC Papers in Press, May 30, 2001, DOI 10.1074/jbc.C100265200

    ABBREVIATIONS

The abbreviations used are: ES, embryonic stem; Gy, gray; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tg, transgene.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.