Kajal Biswas

RNF212 is a dosage-sensitive regulator of crossing-over during mammalian meiosis

Crossing-over ensures accurate chromosome segregation during meiosis, and every pair of chromosomes obtains at least one crossover, even though the majority of recombination sites yield non-crossovers. A putative regulator of crossing-over is RNF212, which is associated with variation in crossover rates in humans. We show that mouse RNF212 is essential for crossing-over, functioning to couple chromosome synapsis to the formation of crossover-specific recombination complexes. Selective localization of RNF212 to a subset of recombination sites is shown to be a key early step in the crossover designation process. RNF212 acts at these sites to stabilize meiosis-specific recombination factors, including the MutSγ complex (MSH4-MSH5). We infer that selective stabilization of key recombination proteins is a fundamental feature of meiotic crossover control. Haploinsufficiency indicates that RNF212 is a limiting factor for crossover control and raises the possibility that human alleles may alter the amount or stability of RNF212 and be risk factors for aneuploid conditions.

Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations

To date, inheritance of a mutant BRCA1 or BRCA2 gene is the best-established indicator of an increased risk of developing breast cancer. Sequence analysis of these genes is being used to identify BRCA1/2 mutation carriers, though these efforts are hampered by the high frequency of variants of unknown clinical significance (VUSs). Functional evaluation of such variants has been restricted due to lack of a physiologically relevant assay. In this study we developed a functional assay using mouse ES cells to study variants of BRCA1. We introduced BAC clones with human wild-type BRCA1 or variants into Brca1-null ES cells and confirmed that only wild-type and a known neutral variant rescued cell lethality. The same neutral variant was also able to rescue embryogenesis in Brca1-null mice. A test of several BRCT domain mutants revealed all to be deleterious, including a VUS. Furthermore, we used this assay to determine the effects of BRCA1 variants on cell cycle regulation, differentiation, and genomic stability. Importantly, we discovered that ES cells rescued by S1497A BRCA1 exhibited significant hypersensitivity after gamma-irradiation. Our results demonstrate that this ES cell-based assay is a powerful and reliable method for analyzing the functional impact of BRCA1 variants, which we believe could be used to determine which patients may require preventative treatments.

Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2cko/ko mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2ko/ko cells. PARP1 deficiency does not restore HR in Brca2ko/ko cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2cko/cko mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Functional evaluation of BRCA2 variants mapping to the PALB2 binding and C-terminal DNA binding domains using a mouse ES cell–based assay

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell–based assay

Biallelic mutations in the human breast cancer susceptibility gene, BRCA2, are associated with Fanconi anemia, implying that some persons who inherit 2 deleterious variants of BRCA2 are able to survive even though it is well established that BRCA2 is indispensable for viability in mice. One such variant, IVS7 + 2T > G, results in premature protein truncation because of skipping of exon 7. Surprisingly, the persons who are either IVS7 + 2T > G homozygous or compound heterozygous are born alive but die of malignancy associated with Fanconi anemia. Using a mouse embryonic stem cell-based functional assay, we found that the IVS7 + 2T > G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2(Δ105)). We demonstrate that BRCA2(Δ105) is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. Evaluation of this transcript in normal and leukemia cells suggests that BRCA2(Δ105) may contribute to the viability of persons inheriting this mutation. In this study, we have also characterized 5 other BRCA2 variants and found 3 of these (p.L2510P, p.R2336H, and p.W2626C) to be deleterious and 2 (p.I2490T and p.K2729N) probably neutral. Such studies are important to understand the functional significance of unclassified BRCA2 variants.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

Significance Recent advances in cancer epigenetics have shown the involvement of epigenetic abnormalities in the initiation and progression of cancer, but their role in cancer-specific aberrant splicing is not clear. The identification of upstream epigenetic regulators of cancer-specific splicing will enable us to therapeutically target aberrant splicing and provide an approach to cancer therapy. Here we have demonstrated a mechanism of intragenic DNA methylation-mediated regulation of alternative splicing by Brother of Regulator of Imprinted Sites (BORIS), which can contribute to breast cancer tumorigenesis by favoring the Warburg effect. The reversal of the Warburg effect was achieved by the inhibition of DNA methylation or down-regulation of BORIS, which may serve as a useful approach to inhibit the growth of breast cancer cells. Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.

BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.Loss of BRCA2 leads to cancer formation. Here, the authors use an insertional mutagenesis approach and identify a multiprotein complex consisting of BRE, USP7 and CDC25A that can support the survival of BRCA2-deficient cells.

Using recombineering to generate point mutations:galK-based positive-negative selection method.

Recombineering is a recombination-based highly efficient method of genetic engineering. It can be used to manipulate the bacterial chromosomal DNA as well as any episomal DNA. Recombineering can be used to insert selectable or nonselectable DNA fragments and subclone DNA fragments without the use of restriction enzymes and also to make precise alterations including single nucleotide changes in the DNA. Here we describe a galactokinase (galK)-based two-step method to generate point mutations in the bacterial artificial chromosome (BAC) insert using the recombineering technology. It takes advantage of the ability to select and also counterselect for the presence of galK.

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the ‘BRCAness’ profile.