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Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

Abstract

Background

Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.

Results

In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.

Conclusions

This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Background

Breast cancer 2 early onset (BRCA2) is a tumor suppressor gene encoding a protein that contributes to homologous recombination (HR)-mediated DNA repair [1]. As BRCA2 plays a crucial role in DNA repair, deleterious mutations in this gene have been reported to be associated with tumorigenesis in dogs and humans [2, 3]. Our previous studies demonstrated that mutation and downregulation of BRCA2 were both associated with canine mammary tumors [4, 5]. We identified loss of heterozygosity as an underlying mechanism by which BRCA2 expression levels are reduced in canine mammary tumors [6]. A recent study has revealed how haploinsufficiency is induced by BRCA2 mutation [7]. Thus, downregulation of BRCA2 is one of the causes of tumorigenesis. In cells, reduced or excess BRCA2 expression creates an imbalance between BRCA2 and RAD51. RAD51 is a DNA recombinase that interacts with BRCA2 and is recruited to DNA damage sites to carry out strand invasion, which is a vital step of homologous recombination. The imbalance between these gene products affects HR activity [8, 9]. These studies suggest that regulation of BRCA2 expression level is important for maintaining the homeostasis of HR.

BRCA2 also contributes to HR in meiosis, and its expression level is higher in the gonads than in other tissues. In germ cells, BRCA2 interacts with RAD51 and DMC1, which are meiosis-specific DNA recombinases, and recruits them to the recombination site to initiate strand exchange [10]. In mice, insufficiency of BRCA2 leads to infertility, mostly because of failures in recombination in germ cells [11]. Thus, the expression level and function of BRCA2 is vital in germ cells, and regulating the BRCA2 expression level is important for meiosis and maintaining DNA integrity. However, in humans, relationships between BRCA2 mutations and fertility have been controversial [12,13,14].

Several mechanisms have been reported to regulate BRCA2 expression levels. For example, the transcription factor nuclear factor-κB transactivates BRCA2 and p53. ZAR1L and PARP1 were reported as repressors of BRCA2 [15,16,17,18]. Several microRNAs have been reported to downregulate BRCA2 expression. The overexpression of these microRNAs have been identified to be associated with tumorigenesis [19, 20]. At the protein level, BRCA2 is ubiquitinated by USP11 to undergo subsequent degradation [21]. Although several factors are involved in regulating BRCA2 expression, the underlying mechanisms still remain unclear. Another mechanism that could be regulating the expression is the production of splicing variants at the 5′ untranslated region (UTR). Alternative splicing has been previously reported to play an important role in the regulation of gene expression [22]. It is thought that the alternative start codons produce unexpected translation products that reduce the levels of primary transcripts. Splicing variants at the 5′ UTR also reduce translational levels by forming a complex and bulky secondary structure that inhibits ribosome sliding.

In this study, we identified novel splicing variants at the 5′ UTR of canine BRCA2 in ovary and testis samples. These splicing variants were also expressed in cultured cells from humans and dogs. The expression levels varied according to the condition of the cells. We also demonstrated that these 5′ UTR splice variants affected the translation efficiency. Surprisingly, the 5′ UTR sequences of canine and human BRCA2 were also shown to play a role in the regulation of transcriptional activity. This is the first study to demonstrate that BRCA2 expression level is regulated by 5′ UTR splicing variants. We also show that the intron 1 region harbored cis-regulatory elements.

Results

A novel exon was identified between canine BRCA2 exons 1 and 2

We identified a novel exon between canine BRCA2 exons 1 and 2 via RT-PCR using samples from two ovaries and one testis (Fig. 1 a). These PCR products were sequenced to determine their splicing patterns (GenBank accession no. LC547963, LC547964, LC547965, LC547966, LC547967 and LC547968). We identified six splicing variants in addition to the annotated transcript (Fig. 1 b). These variants were identified between originally reported exon 1 and exon 2 in 5′ UTR of canine BRCA2 gene and complied with the GU-AG rule (Supplemental Fig. 1). These splice variants resulted in mRNA transcripts that varied in size from 248 bp to 988 bp. We referred to the splicing variants as I-VI, I being the variant that encodes the longest transcript and VI being the shortest. Novel splicing variant I contained 8 possible start codons, which potentially translate short and truncated proteins, and splicing variant VI, the shortest variant, contained 5 possible start codons, whereas the registered sequence does not have a start codon in the 5′ UTR (Supplemental Fig. 1). It is not easy to determine the precise expression level by RT-PCR, but splicing variants I, II, V, VI and registered sequence were the main transcripts (Fig. 1 a). We also tested the expression of these splicing variants in cultured cells derived from dogs. All four cell lines showed the presence of these spliced variants, similar to testis and ovary tissues (Fig. 1 c) and the splicing variants I, II, V, VI and the registered variant showed higher mRNA levels.

Fig. 1
figure1

Identification of novel splicing variants in canine BRCA2 in ovary and testis. a RT-PCR products around the canine BRCA2 intron 1 in two ovary and one testis samples were electrophoresed. Splicing variants, referred to as splicing variant I-VI and registered sequence are indicated. b Diagram of novel splicing variants in canine BRCA2 intron 1. Arrows indicate primers using RT-PCR and nested-PCR. c RT-PCR products from canine cell lines were electrophoresed and splicing variants excluding splicing variant III were detected. RT indicates RT-PCR products and non-RT indicates PCR products without reverse transcription. Arrows indicate primers used in RT-PCR and nested-PCR

Expression pattern and levels of splicing variants in cultured cells were affected by serum starvation

The expression level of BRCA2 is regulated by the cell cycle and the condition of the cells. Thus, we tested the effect of serum starvation, which introduced G1 phase arrest, on cultured cell lines. The expression pattern of splicing variants in CHM-p cells changed due to serum starvation. However, MDCK and CIP-p cells maintained the same expression pattern even after serum starvation (Fig. 2 a). In CNM-p cells, expression levels of splicing variants III and IV and the registered sequence were reduced after serum starvation. In CHM-p cells, levels of splicing variants VI and the registered sequence were reduced after serum starvation (Figs. 1 and 2 a). CNM-p and CHM-p cells also showed relatively increased expression levels of splicing variant II compared to splicing variant I upon serum starvation (Fig. 2 a). In all the four cell lines mentioned above, the expression level of BRCA2 was reduced by serum starvation (Fig. 2 b). The relative ratio of expression levels of the BRCA2 variants with the shortest registered sequence to total BRCA2 was also changed (Fig. 2 c). MDCK and CIP-p cells showed an increased relative expression ratio of registered sequence, but it was decreased in the cases of CNM-p and CHM-p (Fig. 2 c).

Fig. 2
figure2

Serum starvation effects on expression pattern and ratio of BRCA2 5′ UTR splicing variants. a RT-PCR products between canine BRCA2 exon 1 and exon 2 in four canine cell lines with or without 72-h serum starvation were electrophoresed. RT indicates RT-PCR products and non-RT indicates PCR products of the templates without reverse transcription. SV and RS indicate splicing variant and registered sequence, respectively. b Relative expression ratio of registered sequence (ordinary exon 1–2) versus total canine BRCA2 expression (exon 26–27) in four canine cell lines was measured by quantitative PCR. To quantify registered sequence, the exon 1 and 2 spanning primer was used. c Relative expression level of canine BRCA2 normalized by canine RPS18 in four canine cell lines. “**” indicates p < 0.01

Human cultured cell lines also expressed similar splicing variants

Canine tissues and cultured cells expressed splicing variants in canine BRCA2 5′ UTR, and the expression ratio of these variants was regulated by the condition of the cells. We hypothesized that regulation of BRCA2 gene expression via splicing variants could also be present in humans. We tested this using two human derived cell lines, HeLa and 293 T. Splicing variants including the entire intron 1 sequence were expressed in these cell lines (Fig. 3 a and b, GenBank accession no. LC547969). The novel splicing variant contained 9 possible start codons, which potentially translate short and truncated proteins, whereas the registered sequence did not have a start codon in the 5′ UTR (Supplemental Fig. 1). Although the expression pattern of the splicing variants was not changed by serum starvation, the relative ratio of registered sequence and shorter splicing variant was reduced or increased with a slight increase in BRCA2 expression level in HeLa or 293 T cells (Fig. 3 c). We also tested the effect of X-ray irradiation, as some genes have been reported to change the ratio of splicing variants to regulate expression level in response to DNA damage [23, 24]. However, the relative ratio of the registered sequence did not show any drastic change until 60 min after 10 Gy X-ray treatment (Fig. 3 e).

Fig. 3
figure3

Human cell lines, HeLa, and 293 T cells also expressed splicing variants. a RT-PCR products between human BRCA2 exon 1 and exon 2 in HeLa and 293 T cell lines with or without 48-h serum starvation were electrophoresed. RT indicates RT-PCR products and non-RT indicates PCR products using the templates without reverse transcription. Arrows indicates primers using RT-PCR and nested-PCR. “§” indicates PCR products with first PCR primers. b Diagram of novel splicing variants in human BRCA2 intron 1. Arrows indicate primers used in RT-PCR and nested-PCR. c Relative expression ratio of registered sequence (ordinary exon 1–2) versus total human BRCA2 expression (exon 25–26) in HeLa and 293 T cells was measured via quantitative PCR. To quantify the level of the registered sequence, the exon 1 and 2 spanning primer was used. d Relative expression level of human BRCA2 normalized using human RPS18 in HeLa and 293 T cells. e HeLa cells treated with 10 Gy X-rays were harvested at the indicated time points after irradiation. The relative expression ratio of registered sequence (ordinary exon 1–2) versus total human BRCA2 expression (exon 25–26) was measured as in Fig. 3 (c). “**” indicates p < 0.01. “N. S.” indicates not significant

Novel splicing variants of canine and human BRCA2 suppressed translational efficiency

The novel splicing variants of BRCA2 were expressed in canine tissues and both canine and human derived cell lines and these ratios were changed by the conditions in which the cells were maintained. We speculated that these splicing variants regulate the translational efficiency of BRCA2. To evaluate the translational efficiency of BRCA2, luciferase assay with quantitative PCR were employed (Fig. 4 a). To evaluate the translational efficiency, we designed plasmids containing CMV promoter followed by each 5′ UTR variant sequence and luciferase. These constructs might also contribute to the suppression of the transcriptional efficiency. Thus, to avoid this effect, we quantified the expression level of firefly luciferase and the transfection efficiency of the plasmid DNA construct containing firefly luciferase gene and genomic DNA of human BRCA2 exon 27 was determined via quantitative PCR. In canine BRCA2, the splicing variants, I, II, V, VI, and the registered sequence were the major variants in tissues and cell lines. Thus, we evaluated these five variants. Figure 4 b and d shows the relative expression level of firefly luciferase. Unexpectedly, splicing variants I and II of canine BRCA2 and splicing variant I of human BRCA2 suppressed the CMV promoter activity (Fig. 4 b and d). In parallel, luciferase assay was performed to evaluate the translational activity. The values of the transcriptional activity were used to normalize the relative translational activity. The splicing variants of canine BRCA2 excluding splicing variant I and splicing variant I of human BRCA2 were found to be associated with decreased translational activity (Fig. 4 c and e).

Fig. 4
figure4

Effect of novel splicing variants on transcription/translation activity in cells. a Schematic diagram depicting how the transcriptional and translational activity were measured. Firefly luciferase expression vectors with each splicing variant and Renilla luciferase expression vector were transfected to HeLa cells followed by DNA, mRNA, and protein extraction after 48 h. The relative expression level of firefly luciferase mRNA was measured through quantitative PCR normalized with the house-keeping gene human RPS18 and the transfection efficiency by genomic luciferase and human BRCA2 exon 27 region from DNA samples. To determine the efficiency of translation activity, firefly and Renilla luciferase activity were detected using dual-luciferase reporter assay normalized by transcriptional activity. b Relative transcriptional efficiency of canine BRCA2 splicing variants containing luciferase vector. c Relative translational efficiency of canine BRCA2 splicing variants containing luciferase vector. d Relative transcriptional efficiency of human BRCA2 splicing variants containing luciferase vector. c Relative translational efficiency of human BRCA2 splicing variants containing luciferase vector. “**” indicates p < 0.01

Intron 1 of canine and human BRCA2 also suppressed BRCA2 promoter activity

Novel splicing variants of canine and human BRCA2 suppressed CMV promoter activity in addition to the translational suppression. Thus, we next tested the effect of the DNA sequence of various splicing variants on BRCA2 promoter activity. Prior to this, canine and human BRCA2 intron 1 regions were compared to find conserved DNA sequences, because the novel exon region identified in intron 1 was different from the registered sequence and conserved sequence had the greatest potential to be a silencer element (Fig. 5). Three conserved regions were identified in canine and human BRCA2. A conserved 15 bp 5′-terminal region was located in the BRCA2 promoter. The other two regions, human BRCA2 + 460 − + 617 bp and + 732 − + 942 bp, were also found to be conserved within canine BRCA2 intron 1. Thus, we expected these two conserved DNA sequences to have a silencer element. Because canine BRCA2 splicing variant II, which did not have 3′-terminal 95 bp of intron 1, still suppressed the effect of CMV promoter activity, thus the sequence around + 460 − + 841 bp in human BRCA2 (corresponding + 413 − + 787 bp in canine BRCA2) should also be associated with silencer elements. To avoid translational effects, the human BRCA2 intron 1 region without promoter sequence (− 187 − + 310 bp) was inserted upstream of human BRCA2 promoter (Fig. 6 a). As expected, + 443–843 bp region in human BRCA2 decreased the promoter activity similar to human BRCA2 intron 1 region (+ 311–942 bp). Next, we attempted to identify where the silencer element was located within the 500 bp sequence. Sequential deletion mutants in 5′ and 3′ ends were compared with the promoter activity (Fig. 6 b and c). The + 743 − + 842 bp region contained a cis-regulatory silencer element (Fig. 6 c). The region of + 443 − + 542 bp also contained a silencer, as the presence of this region showed significantly reduced promoter activity than that of the + 543 − + 842 bp region (Fig. 6 c) and the + 443 − + 542 bp region tended to reduce the promoter activity but it was not significant (Fig. 6 b; p = 0.0295, by Holm’s method; p value < 0.0125 was considered significant.). Unexpectedly, the region around + 643 − + 742 bp harbored an enhancer element, and the + 543 − + 642 bp region contained a silencer element (compare Fig. 6 c to Fig. 6 b). Thus, we speculated that cis-regulatory elements could be separated in some constructs. To study this region in detail, three distinct constructs were tested (Fig. 6 d). As we expected, the + 593 − + 692 bp region contained a silencer element. However, surprisingly, the + 543 − + 642 bp region acted as an enhancer element. To identify the enhancer elements around + 742 bp in intron 1, we also analyzed the + 693 − + 792 bp region (Fig. 6 e). This region showed slightly decreased BRCA2 promoter activity. Thus, we concluded that the silencer and enhancer elements were too close to be separated, and the effect of the silencer element in this region was stronger than the enhancer element.

Fig. 5
figure5

Alignment of canine and human BRCA2 intron 1. Canine and human BRCA2 intron 1 were aligned by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Three regions are conserved between canine and human BRCA2 intron 1. The lower panel shows a dot plot view of the alignment

Fig. 6
figure6

Effect of human BRCA2 intron 1 region on transcription activity. Human BRCA2 intron 1 region was inserted upstream of human BRCA2 promoter region (− 187 − + 310 bp) to measure the transcriptional activity using dual-luciferase reporter assay. a The promoter activities of the entire intron 1 region without promoter (+ 311 − + 942 bp) and conserved central 500 bp (+ 443 − + 842 bp) were measured. The promoter activities of sequential 100 bp deletion from 3′ end (b) and 5′ end (c) were compared. d and e To analyze in more detail the cis-regulatory elements in human BRCA2 intron 1, the indicated 100 bp of human BRCA2 intron 1 was inserted upstream of human BRCA2 promoter and compared. “*” indicates p < 0.05 compared with the construct that only had the BRCA2 promoter. “N. S.” indicates not significant.

Discussion

BRCA2 expression levels must be tightly regulated, in particular to maintain a balance between the expression level of BRCA2 and RAD51, which is required for effective HR [9, 25]. Here, we identified a new translation regulation mechanism in both canine and human BRCA2, which is mediated by splicing variants at the 5′ UTR that have been reported to usually regulate gene expression [22]. All canine BRCA2 splicing variants excluding splicing variant I and human BRCA2 splicing variant I decreased translational efficiency.

The expression ratios of these splicing variants were altered by the condition of the cells. In CNM-p and CHM-p canine cultured cells, the ratio of transcripts without the intron 1 region compared to total BRCA2 was decreased after serum starvation, which induced G1 phase arrest. Especially in CNH-p and CHM-p cells, the splicing variant pattern was also shifted to favor the long form after serum starvation. Splicing variants containing the intron 1 region, excluding canine splicing variant I, showed decreased translational activity. Thus, in these cells, BRCA2 protein levels were decreased via suppression of translation. MDCK and CIP-p canine cells demonstrated an increased ratio of splicing variants without the intron 1 region after serum starvation. In addition to this, the decreased level of BRCA2 mRNA in these cells was relatively not as significant as in case of CNM-p and CHM-p cells. Thus, these cells seemed to maintain the BRCA2 protein level after serum starvation. Like canine cell lines, there were two types of responses in human cells. In HeLa cells, the ratio of transcripts without the intron 1 region versus total BRCA2 was decreased, whereas it increased in 293 T cells. Splicing variant I of human BRCA2 showed decreased translational activity. Thus, in HeLa cells, the BRCA2 protein level was decreased via suppression of translation, but not increased in 293 T cells. However, why there is a difference in response is not clear. We speculated that the sensitivity to the serum starvation was associated with BRCA2 expression level and splicing variant ratio, because CNM-p and CHM-p cells were more sensitive to serum starvation than MDCK cells (data not shown). We also speculated that these splicing variants contributed to the protein expression after X-ray irradiation, as greater amounts of transcripts of splicing variants were induced in response to DNA damage [23, 24]. However, the ratio of splicing variants was not altered by irradiation, thus the novel splicing variant of BRCA2 at 5′ UTR was not affected by DNA damage.

The mechanisms underlying the suppression of expression by these novel splicing variants are unknown, but there are two possibilities based on the reports on the regulation of other genes [22]. First, regulation may occur through the upstream start codon in the 5′ UTR. The upstream start codons translate short or truncated proteins and prevent the production of the main open reading frame products [22]. Each novel splicing variant of canine BRCA2 possesses 5–8 start codons which potentially translate short peptides or truncated proteins. As the reference sequence does not contain a start codon in the 5′ UTR, these upstream start codons in the 5′ UTR potentially prevent translation efficiency of the primary open reading frame. The second possibility involves the RNA secondary structure. Both novel splicing variants showed complex and bulky structures according to a secondary RNA structure prediction program. This type of structure obstructs ribosome sliding and scanning. Some genes are regulated by the RNA secondary structure of splicing variants at the 5′ UTR [22]. Although splicing variant I of canine BRCA2 still possessed similar translational activity compared to the registered sequence, canine and human BRCA2 are potentially regulated by these mechanisms.

The sequence of the splicing variants affected transcription efficiency as well as translation. Thus, canine and human intron 1 regions in the genomic DNA act as cis-regulatory elements. A few studies have described BRCA2 promoter silencer elements, but this is the first study to show that this region, which is a part of the BRCA2 intron 1, functions as cis-regulatory elements [15,16,17, 26]. First, we expected that there had been only a silencer element in intron 1, but our thorough analysis indicated that this region consisted with at least three silencer elements and two enhancer elements. Supporting this notion, in human BRCA2 intron 1, accumulation of histone H3K27Ac, which indicates the presence of active promoter and enhancer regions, were reported by the UCSC genome browser (https://genome.ucsc.edu). We did not investigate which trans-regulatory elements interact with this novel cis-regulatory element. This point will be the next important research issue to understand how BRCA2 expression is regulated.

Although BRCA2 was identified more than a twenty-five year ago and many studies of this gene have been reported in humans, canine and mice, the regulatory system identified in this study has not been previously reported. Both canine and human BRCA2 had similar splicing variants, thus it seemed that mammalian BRCA2 employs splicing variants at the 5′ UTR as an expression regulation system. Parts of the intron 1 region were conserved between human and canine, thus transcriptional regulation by the novel cis-regulatory element also seemed to be conserved in mammals. These points are interesting when considering the evolution of BRCA2.

Conclusions

In this study, we identified novel splicing variants at the 5′ UTR of canine and human BRCA2 in canine ovary and testis and canine and human cell lines. These splicing variants reduced the translation efficiency of the gene. The expression ratio of splicing variants was altered depending on the conditions in which the cells were maintained. Unexpectedly, the intron 1 sequence in BRCA2 also suppressed transcription efficiency. This led to the identification of silencer and enhancer cis-regulatory elements in BRCA2 intron 1 region. Although further studies are needed to determine how the splicing variants regulate translation and how novel cis-regulatory elements affect transcription, our data suggests that the BRCA2 expression level was regulated by the novel splicing variants at the 5′ UTR and cis-regulatory elements in intron 1 in canine and human.

Methods

Cell culture

All cultured cells were grown in 100 mm plastic dishes containing 10 mL of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under humidified 5% CO2–95% air.

RNA extraction for reverse-transcription PCR

Two ovary and one testis samples from three 1-year-old mixed breed dogs were obtained from the Honda Animal Hospital. This sampling was done as part of contraception or castration under general anesthesia by veterinarians. These tissue samples in RNAlater solution (Life Technologies, Carlsbad, CA, USA) and all cultured cell pellets were stored at − 80 °C until they were processed for RNA extraction using TRIzol RNA Isolation Reagents (Life Technologies, Carlsbad, CA, USA) as previously described [27]. All experimental procedures were approved by and conducted in accordance with the Guidelines for Institutional Laboratory Animal Care and Use of the School of Veterinary Medicine at Kitasato University, Japan (Approval Number: 11–065).

Reverse-transcription-PCR and nested PCR

Reverse-transcription of RNA were performed using SuperScript™ IV VILO™ Master Mix (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. To amplify canine and human BRCA2, each reaction mixture contained 100 ng of genomic DNA as a template, and 300 nM forward and reverse primers (Table 1), and KOD One PCR master mix (Toyobo, Osaka, Japan) was used with a total volume of 100 μL. PCR conditions included one cycle of initial denaturation for 10 s at 98 °C, followed by 25 cycles with 10 s of denaturation at 98 °C, 25 s of annealing and elongation at 68 °C, and a final extension step of 1 min at 68 °C. The PCR products were diluted 100–2000 times and PCR was performed with same conditions using nested-PCR primer sets. The PCR products were sequenced to identify the splicing variants.

Table 1 Base sequences of primers used in this study

Luciferase assay

Approximately 2 × 105 HeLa cells were transiently co-transfected with 20 ng canine or human BRCA2 5′ UTR and firefly luciferase cloned pcDNA3 (Invitrogen) or pGL4 (Promega) vectors and 10 ng pRL-TK (Promega) using FuGENE HD Transfection Reagent (Roche Diagnostics, Basel, Switzerland). Forty-eight hours after transfection, the cells were lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions. Transfection efficiency was normalized by measuring Renilla luciferase activity.

Quantitative real-time PCR

RNA was extracted from the transfected cells using the CellAmp™ Direct RNA Prep Kit for RT-PCR (Real Time) (Takara, Japan), and cDNA synthesis was performed as described above. Genomic DNA was also prepared using a Gentra Puregene Cell Kit (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction (qPCR) was carried out in a StepOnePlus Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) using a KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and 200 nM of each primer (Table 1). The PCR cycling conditions were as follows: an initial denaturation of 95 °C for 20 s followed by 40 PCR cycles of 95 °C for 3 s and 60 °C for 30 s. Melting curves were generated at the end of each real-time PCR run to ensure that a single specific product was amplified. Each sample was evaluated in triplicate. The housekeeping gene canine or human RPS18 was used as an internal reference for normalization. The human BRCA2 exon 27 region and firefly luciferase from genomic DNA samples were used to normalize the transfection efficiency.

Treatment with ionizing radiation

Approximately 2 × 105 HeLa cells were seeded into 4-well plates. After 24 h, irradiation of HeLa cells in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum was carried out in a MX-80Labo (mediXtec Japan, Japan) for a total dose of 10 Gy (dose rate; 0.67 Gy/min) at room temperature. Irradiated cells were incubated at 37 °C under humidified 5% CO2–95% air and after the indicated time, the cells were harvested and the RNA was extracted.

Statistical analysis

To compare two groups, statistical analyses were performed using an F test followed by Student’s t-test. To compare more than three groups, one-way analysis of variance with Dunnett’s test or F test followed by Student’s t-test correcting with Holm’s method was performed.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BRCA2:

Brest cancer 2, early onset

HR:

Homologous recombination

RAD51:

RAD51 recombinase

DMC1:

DNA meiotic recombinase 1

ZAR1L:

Zygote arrest 1 like

PARP1:

Poly (ADP-ribose) polymerase 1

USP11:

Ubiquitin specific peptidase 11

RT-PCR:

Reverse-transcription-polymerase chain reaction

PCR:

Polymerase chain reaction

CMV:

Cytomegalovirus

H3K27Ac:

Histone H3 acetylated lysine 27

UCSC:

University of California Santa Cruz

References

  1. 1.

    Fradet-Turcotte A, Sitz J, Grapton D, Orthwein A. BRCA2 functions: from DNA repair to replication fork stabilization. Endocr Relat Cancer. 2016;23:T1–T17.

    CAS  Article  Google Scholar 

  2. 2.

    Rivera P, Melin M, Biagi T, Fall T, Haggstrom J, Lindblad-Toh K, et al. Mammary tumor development in dogs is associated with BRCA1 and BRCA2. Cancer Res. 2009;69:8770–4.

    CAS  Article  Google Scholar 

  3. 3.

    Scully R, Livingston DM. In search of the tumour-suppressor functions of BRCA1 and BRCA2. Nature Nature Publishing Group. 2000;408:429–32.

    CAS  Google Scholar 

  4. 4.

    Yoshikawa Y, Morimatsu M, Ochiai K, Ishiguro-Oonuma T, Wada S, Orino K, et al. Reduced canine BRCA2 expression levels in mammary gland tumors. BMC Vet Res BioMed Central. 2015;11:159.

    Article  Google Scholar 

  5. 5.

    Yoshikawa Y, Morimatsu M, Ochiai K, Nagano M, Yamane Y, Tomizawa N, et al. Analysis of genetic variations in the exon 27 region of the canine BRCA2 locus. J Vet Med Sci. 2005;67:1013–7.

    CAS  Article  Google Scholar 

  6. 6.

    Yoshikawa Y, Morimatsu M, Ochiai K, Nagano M, Tomioka Y, Sasaki N, et al. Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors. Am J Vet Res. 2008;69:1323–8.

    CAS  Article  Google Scholar 

  7. 7.

    Tan SLW, Chadha S, Liu Y, Gabasova E, Perera D, Ahmed K, et al. A class of environmental and endogenous toxins induces BRCA2 Haploinsufficiency and genome instability. Cell Elsevier Inc. 2017;169:1105–15.

    CAS  Google Scholar 

  8. 8.

    Magwood AC, Malysewich MJ, Cealic I, Mundia MM, Knapp J, Baker MD. Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing. DNA Repair. 2013;12:1122–33.

    CAS  Article  Google Scholar 

  9. 9.

    Arnold K, Kim M-K, Frerk K, Edler L, Savelyeva L, Schmezer P, et al. Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair. Cancer Lett. 2006;243:90–100.

    CAS  Article  Google Scholar 

  10. 10.

    Martinez JS, von Nicolai C, Kim T, Ehlén Å, Mazin AV, Kowalczykowski SC, et al. BRCA2 regulates DMC1-mediated recombination through the BRC repeats. Proc Natl Acad Sci USA. National Academy of Sciences. 2016;113:3515–20.

    CAS  Article  Google Scholar 

  11. 11.

    Sharan SK, Pyle A, Coppola V, Babus J, Swaminathan S, Benedict J, et al. BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development. 2004;131:131–42.

    CAS  Article  Google Scholar 

  12. 12.

    Daum H, Peretz T, Laufer N. BRCA mutations and reproduction. Fertil Steril. 2018;109:33–8.

    CAS  Article  Google Scholar 

  13. 13.

    Lin W, Titus S, Moy F, Ginsburg ES, Oktay K. Ovarian aging in women with BRCA Germline mutations. J Clin Endocrinol Metab. 2017;102:3839–47.

    Article  Google Scholar 

  14. 14.

    Zhoucun A, Zhang S, Yang Y, Ma Y, Zhang W, Lin L. The common variant N372H in BRCA2 gene may be associated with idiopathic male infertility with azoospermia or severe oligozoospermia. Eur J Obstet Gynecol Reprod Biol. 2006;124:61–4.

    CAS  Article  Google Scholar 

  15. 15.

    Wu K, Jiang S-W, Couch FJ. p53 mediates repression of the BRCA2 promoter and down-regulation of BRCA2 mRNA and protein levels in response to DNA damage. J Biol Chem. 2003;278:15652–60.

    CAS  Article  Google Scholar 

  16. 16.

    Misra S, Sharma S, Agarwal A, Khedkar SV, Tripathi MK, Mittal MK, et al. Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells. Mol Cancer. 2010;9:50.

    Article  Google Scholar 

  17. 17.

    Wang J, Bian C, Li J, Couch FJ, Wu K, Zhao RC. Poly (ADP-ribose) polymerase-1 down-regulates BRCA2 expression through the BRCA2 promoter. J Biol Chem. 2008;283:36249–56.

    CAS  Article  Google Scholar 

  18. 18.

    Wu K, Jiang SW, Thangaraju M, Wu G, Couch FJ. Induction of the BRCA2 promoter by nuclear factor-kappa B. J Biol Chem. 2000;275:35548–56.

    CAS  Article  Google Scholar 

  19. 19.

    Choi YE, Pan Y, Park E, Konstantinopoulos P, De S, D'Andrea A, et al. MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability. Elife. 2014;3:e02445.

    Article  Google Scholar 

  20. 20.

    Song L, Dai T, Xie Y, Wang C, Lin C, Wu Z, et al. Up-regulation of miR-1245 by c-myc targets BRCA2 and impairs DNA repair. J Mol Cell Biol. 2012;4:108–17.

    CAS  Article  Google Scholar 

  21. 21.

    Schoenfeld AR, Apgar S, Dolios G, Wang R, Aaronson SA. BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage. Mol Cell Biol. 2004;24:7444–55.

    CAS  Article  Google Scholar 

  22. 22.

    Hinnebusch AG, Ivanov IP, Sonenberg N. Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science. Am Assoc Advancement Sci. 2016;352:1413–6.

    CAS  Google Scholar 

  23. 23.

    Shkreta L, Chabot B. The RNA splicing response to DNA damage. Biomolecules. 2015;5:2935–77.

    CAS  Article  Google Scholar 

  24. 24.

    Sprung CN, Li J, Hovan D, McKay MJ, Forrester HB. Alternative Transcript Initiation and Splicing as a Response to DNA Damage. Bernhard EJ, editor. PLoS ONE. Public Libr Sci. 2011;6:e25758–20.

    CAS  Google Scholar 

  25. 25.

    Magwood AC, Mundia MM, Baker MD. High levels of wild-type BRCA2 suppress homologous recombination. J Mol Biol. 2012;421:38–53.

  26. 26.

    Tripathi MK, Misra S, Khedkar SV, Hamilton N, Irvin-Wilson C, Sharan C, et al. Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells. J Biol Chem. 2005;280:17163–71.

    CAS  Article  Google Scholar 

  27. 27.

    Yoshikawa Y, Morimatsu M, Ochiai K, Okuda K, Taoda T, Chikazawa S, et al. Establishment of a PCR analysis method for canine BRCA2. BMC Res Notes. 2012;5:173.

    CAS  Article  Google Scholar 

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Acknowledgements

We sincerely thank Dr. Takayuki Nakagawa and Dr. Nobuo Sasaki (The University of Tokyo) for providing the canine mammary gland tumor cell lines used in this study.

Funding

This work was supported in part by a Kitasato University Research Grant for Young Researchers, the Sasakawa Scientific Research Grant from The Japan Science Society, and Grants-in-Aid for Young Scientists (B) [grant numbers 16 K18817 and 20 K06417] from the Japan Society for the Promotion of Science. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.

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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by YY, KS, and HK. The first draft of the manuscript was written by YY, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yasunaga Yoshikawa.

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Collection of ovary and testis samples were approved by and conducted in accordance with the Guidelines for Institutional Laboratory Animal Care and Use of the School of Veterinary Medicine at Kitasato University, Japan (Approval Number: 11–065).

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Not applicable.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Fig. S1.

Messenger RNA base sequences encoded by exon 1-intron 1-exon 2 region of canine and human BRCA2. Bold characters indicate possible start codons and bold AUG with box indicates actual start sites in exon 2. Underlined characters indicate the GU-AG sites for splicing.

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Yoshikawa, Y., Kozuma, H., Morimatsu, M. et al. Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2. BMC Mol and Cell Biol 22, 2 (2021). https://0-doi-org.brum.beds.ac.uk/10.1186/s12860-020-00336-4

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Keywords

  • BRCA2
  • Expression level
  • Splicing variant
  • Translation efficiency
  • 5′ untranslated region