| pmid | 10373512 | ||||||
|---|---|---|---|---|---|---|---|
| title | Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals. | ||||||
| authors |
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| journal | Mol Cell Biol | ||||||
| year | 1999 | ||||||
| full_text_available | true | ||||||
| full_text_extraction_method | html | ||||||
| pmcid | PMC84261 | ||||||
| doi | 10.1128/MCB.19.7.4633 |
Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.
Authors: Marston NJ, Richards WJ, Hughes D, Bertwistle D, Marshall CJ, Ashworth A Journal: Mol Cell Biol (1999) DOI: 10.1128/MCB.19.7.4633 PMC: PMC84261
- Mol Cell Biol. 1999 Jul;19(7):4633-42. doi: 10.1128/MCB.19.7.4633.
Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.
Marston NJ(1), Richards WJ, Hughes D, Bertwistle D, Marshall CJ, Ashworth A.
Author information: (1)Section of Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom SW3 6JB.
Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.
DOI: 10.1128/MCB.19.7.4633 PMCID: PMC84261 PMID: 10373512 [Indexed for MEDLINE]
Abstract
Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2 , we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae . Yeast strains in which these DSS1 -like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.
DISCUSSION
The frequently observed loss of the wild-type copy of BRCA2 in breast tumors in individuals heterozygous for a loss-of-function mutation suggests that the gene encodes a tumor suppressor ( 3 , 9 ). Potential functions that have been attributed to BRCA2 include a role in transcriptional regulation and an involvement in DNA repair via interaction with RAD51 ( 30 , 42 ). However, further information is needed to clarify such roles and to identify additional functions of this very large protein. Using the yeast two-hybrid system with fragments of human BRCA2 as bait, we have found an interaction between a C-terminal portion of BRCA2 and the human DSS1 protein. The validity of this association was supported by demonstration of an association using a mammalian two-hybrid system, and this interaction was reproduced in mammalian cells transiently transfected with cDNA constructs. Furthermore, an endogenous complex including the two proteins was identified in breast cancer cell line MCF7. The C-terminal section of BRCA2 (amino acids 2472 to 2957) which interacts with DSS1 in yeast is distinct from regions of the protein involved in the association with RAD51 ( 42 , 52 ). This fragment of BRCA2 represents a region of higher conservation between human and mouse sequences than the open reading frame as a whole, with 77% identity at the amino acid level compared with 59% overall ( 11 ). This suggests an important function of this region which is conserved. Attempts to further localize the BRCA2 residues involved in the interaction with DSS1 by using sections of amino acids 2472 to 2957 of BRCA2 in yeast two-hybrid analyses with full-length DSS1 failed to identify any shorter fragments which retained the association (data not shown), suggesting that there may be multiple independent determinants in this region for the interaction. The interaction between BRCA2 and DSS1 is likely to be direct, due to the demonstration in the yeast two-hybrid system, but the requirement of a cofactor cannot be ruled out in the absence of the use of purified proteins in vitro.
Numerous tumor-associated frameshift mutations in BRCA2 are located in or upstream of the sequence encoding the region which interacts with DSS1, resulting in truncations which delete all or part of this region of BRCA2 ( 12 , 17 , 48 , 53 ). Such mutations include the 6174delT mutation associated with Ashkenazi Jewish lineages which results in a truncation of the BRCA2 protein prior to the eighth BRC repeat ( 34 , 49 ). The frequency of reported missense polymorphisms within amino acids 2472 to 2957 of BRCA2 is very low ( 12 , 48 ). However, a few frameshift mutations which would result in truncation of BRCA2 between the DSS1-interacting region and the site of the C-terminal polymorphic stop codon 3326 suggest that loss of a more C-terminal function is critical to loss of tumor suppression activity ( 12 , 17 , 28 , 36 ). The human DSS1 gene is located at chromosome 7q21, and frequent loss of heterozygosity at 7q is seen in a range of cancers, suggesting the presence of a tumor suppressor gene in this region. However, although 7q21 rearrangements have been identified in tumors, the minimum commonly deleted region appears to be around 7q31 ( 1 , 25 ).
DSS1 is one of three candidates identified on 7q21.3-q22.1 in a search for genes involved in an autosomal dominant form of the heterogeneous limb developmental disorder SHFM, which is characterized by missing or fused digits ( 13 , 22 , 40 ). Chromosomal rearrangement breakpoints at the SHFM1 locus on 7q from a subset of affected patients defined a critical region containing the DSS1 gene and two Distal-less homeobox genes, DLX5 and DLX6 , all of which are expressed in the mouse in patterns which could implicate these genes in limb and facial development ( 13 , 46 ). The human and mouse DSS1 genes encode identical 70-amino-acid highly acidic proteins of unknown function ( 13 ). The absolute conservation of human and mouse DSS1 and the identification here of highly conserved yeast orthologues, both in S. pombe and in S. cerevisiae , suggests that DSS1 has an important cellular function in eukaryotes.
To understand the significance of the interaction of BRCA2 with DSS1, we wished to gain some information as to the normal cellular role of DSS1. The yeasts S. cerevisiae and S. pombe were used as model systems. Deletions of the DSS1 -like open reading frames in the fission and budding yeasts were made in order to investigate the role of DSS1 in these organisms. The deletion of DSS1 was not lethal to either yeast, but the gene appeared to be required for normal growth control. S. pombe Δdss1 strains grew more slowly than wild-type cells, particularly at reduced or elevated temperatures, exhibiting a cell cycle delay with cells gaining a considerably increased length before undergoing division. Analyses of S. pombe Δdss1 cells by flow cytometry and confocal microscopy revealed that the mutant cells have a defect in completion of cell division which is more pronounced at elevated temperature, leading to an accumulation of cells with greater than 2C DNA content. S. cerevisiae Δdss1 strains were also sensitive to elevated temperature. No BRCA2 orthologue has been identified in either S. pombe or S. cerevisiae . However this does not preclude the possibility that some other gene serves a similar function in yeast. The conservation of function of DSS1 is strongly suggested by the rescue of S. pombe Δdss1 phenotypes by overexpression of human DSS1 . Given the potential role of BRCA2 in DNA repair ( 35 , 42 ), we have measured the sensitivity of Δdss1 yeast to ionizing radiation. Preliminary experiments suggest that although deleted S. pombe cells appear to be marginally more sensitive than wild-type strains, the effect is much less than would be expected for a gene directly involved in DNA repair ( 43 ). The identification of yeast DSS1 should provide a useful system to analyze the function of the gene and should help elucidate its relationship to the cellular role of BRCA2.
Both DSS1 and BRCA2 genes appear to be ubiquitously expressed in mouse tissues, as assayed by reverse transcription-PCR and Northern blotting ( 11 , 13 , 27a , 37 ). The cellular localization was investigated; expression of DSS1 protein in MCF7 cells appears to be growth regulated, with very low amounts detected in serum-starved cells and expression elevated rapidly after refeeding with serum. BRCA2 expression is also very low in serum-starved MCF7 cells but accumulates only as the cells reach the G 1 /S boundary after serum stimulation ( 4 ). The maintenance of high levels of DSS1 after addition of serum to culture medium means that both proteins may be expressed together in the nucleus, particularly during S phase. DSS1 has no obvious nuclear localization signal and so may be transported and anchored in the nucleus by interaction with another protein, conceivably BRCA2.
Since submission of this report, the S. cerevisiae DSS1 gene has been described elsewhere ( 24 ). The gene is implicated in exocytosis and pseudohyphal differentiation in S. cerevisiae . The relationship between these two processes in S. cerevisiae and the phenomena that we observe in Δdss1 S. pombe is not clear but may suggest that DSS1 is involved in multiple cellular processes.
The functions of the BRCA2 gene are unclear, although there is evidence for roles in DNA repair and transcriptional regulation ( 3 ). Here we show that BRCA2 associates in yeast and mammalian cells with the product of the previously described gene DSS1 . This protein appears to be required in yeast for proper completion of cell division, providing a link between BRCA2 and the cell cycle. Whether DSS1 is involved in the etiology of tumors in women carrying mutations in BRCA2 is under investigation.