DNA double strand breaks (DSBs) are among the most toxic DNA lesions and can be repaired accurately through homologous recombination (HR). HR requires processing of the DNA ends by nucleases (DNA end resection) in order to generate the required single-stranded DNA (ssDNA) regions. The SWI/SNF chromatin remodelers are 10–15 subunit complexes that contain one ATPase (BRG1 or BRM). Multiple subunits of these complexes have recently been identified as a novel family of tumor suppressors. These complexes are capable of remodeling chromatin by pushing nucleosomes along the DNA. More recent studies have identified these chromatin remodelers as important factors in DNA repair. Using the DR-U2OS reporter system, we show that the down regulation of BRG1 significantly reduces HR efficiency, while BRM has a minor effect. Inactivation of BRG1 impairs DSB repair and results in a defect in DNA end resection, as measured by the amount of BrdU-containing ssDNA generated after DNA damage. Inactivation of BRG1 also impairs the activation of the ATR kinase, reduces the levels of chromatin-bound RPA, and reduces the number of RPA and RAD51 foci after DNA damage. This defect in DNA end resection is explained by the defective recruitment of GFP-CtIP to laser-induced DSBs in the absence of BRG1. Importantly, we show that BRG1 reduces nucleosome density at DSBs. Finally, inactivation of BRG1 renders cells sensitive to anti-cancer drugs that induce DSBs. This study identifies BRG1 as an important factor for HR, which suggests that BRG1-mutated cancers have a DNA repair vulnerability that can be exploited therapeutically.
The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53’s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.
Cancer Drug Resistance is an open access journal, focusing on pharmacological aspects of drug resistance and its reversal, molecular mechanisms of drug resistance and drug classes, etc. Both clinical and experimental aspects of drug resistance in cancer are included.
Summary Analysis of the N ‐ethyl‐ N ‐nitrosourea ( ENU )‐induced repro42 mutation previously identified spermatogenesis associated 22 ( Spata22 ) as a gene required for meiotic progression and fertility in both male and female mice, but its specific contribution to the process was unclear. Here, we report on a novel, null allele of Spata22 ( Spata22 Gt ) and confirm its requirement for germ cell development. Similar to repro42 mutant mice, histological and mating analyses indicate that gametogenesis is profoundly affected in Spata22 Gt/Gt males and females, resulting in infertility. Cytological examination confirms that germ cells do not progress beyond zygonema and meiotic arrest is linked to impairment of both synapsis and DNA repair. Analysis of SPATA 22 distribution reveals that it localizes to foci associated with meiotic chromosomes during prophase I and that the number of foci peaks at zygonema; there are also more SPATA 22 foci in oocytes than in spermatocytes. Furthermore, SPATA 22 co‐localizes with a number of proteins involved in meiotic recombination, including RAD 51, DMC 1, and MLH 1, and is present until mid‐pachynema, suggesting a role in resolution of recombination intermediates. In fact, SPATA 22 co‐localizes with MLH 1 in more than 20% of foci at pachynema. Analysis of Spata22 Gt/Gt meiocytes confirms that SPATA 22 is required for localization of MEIOB but not RPA (two proteins known to interact with SPATA 22), and immunoblotting corroborates that production of MEIOB is indeed decreased in the absence of SPATA 22. Together, these data suggest that SPATA 22 is required for both meiotic recombination and synapsis during meiosis in mice.
Switch/Sucrose non-fermenting (SWI/SNF) chromatin remodelers hydrolyze ATP to push and slide nucleosomes along the DNA thus modulating access to various genomic loci. These complexes are the most frequently mutated epigenetic regulators in human cancers. SWI/SNF complexes are well known for their function in transcription regulation, but more recent work has uncovered a role for these complexes in the repair of DNA double strand breaks (DSBs). As radiotherapy and most chemotherapeutic agents kill cancer cells by inducing double strand breaks, by identifying a role for these complexes in double strand break repair we are also identifying a DNA repair vulnerability that can be exploited therapeutically in the treatment of SWI/SNF-mutated cancers. In this review we summarize work describing the function of various SWI/SNF subunits in the repair of double strand breaks with a focus on homologous recombination repair and discuss the implication for the treatment of cancers with SWI/SNF mutations.
Abstract The tumor repressor gene, p53, is involved in a variety of cellular activities ranging from stress, differentiation and cell cycle regulation. In our previous studies we have shown p53’s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression while the same is absent in p53 null mice. This deficiency of p53 has also been shown to produce more osteosarcomas than a complete loss of p53. This suggests that the presence of p53 is able to modify the environment within pre osteoblasts based on its ability to regulate key bone specific genes. In the present study we compared changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels in C2C12 cells capable of both myoblast and osteoblast differentiation transiently, and compared the changes in gene expression of candidate genes to cells with stable p53 knockdown. Using a PCR array to assay p53 target genes, we have found differential expression profiles when comparing stable versus transient knockdown. As expected several of genes that were profoundly affected after transient p53 loss were related to apoptosis and cell cycle regulation. Stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles suggesting that long term effect of p53 loss affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were also validated by measuring promoter activities of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression profoundly affecting normal tissue physiology than its acute loss. Citation Format: Oliver Couture, Eric Lombardi, Kendra Davis, Emily Hays, Nalini Chandar. Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5438. doi:10.1158/1538-7445.AM2013-5438
